Monoclonal antibodies to growth and differentiation factor 15 (GDF-15), and uses thereof for treating cancer cachexia and cancer

ABSTRACT

The present invention relates to monoclonal anti-human-GDF-15 antibodies. The antibodies include chimeric antibodies and humanized antibodies. The invention also relates to monoclonal anti-human-GDF-15 antibodies including murine antibodies, chimeric antibodies and humanized antibodies for use in methods for the treatment of cancer cachexia and also for the treatment of cancer. The invention also provides pharmaceutical compositions, kits, methods and uses and cell lines capable of producing the monoclonal antibodies of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/128,604, filed Sep. 23, 2016, now abandoned, which is a 35 U.S.C. §371 filing of International Patent Application No. PCT/EP2015/056654,filed Mar. 26, 2015, which claims priority to Great Britain PatentApplication Nos. 1405477.9 and 1405475.3, both filed Mar. 26, 2014. Eachof the aforementioned applications is hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention relates to monoclonal anti-human-GDF-15antibodies, pharmaceutical compositions, kits, methods and uses and thecell lines capable of producing the monoclonal antibodies describedherein. The present invention further relates to antibodies to humanGDF-15 capable of inhibiting cancer growth, treating cancer-inducedweight loss and cancer cachexia.

BACKGROUND

To date, many cancers are still areas of unmet medical needs, andaccordingly, means to more effectively treat cancer, and to treat cancerin a broader range of cancers are needed.

In addition to the suffering caused by the cancer itself, many patientssuffer from cancer cachexia, a medical condition triggered by the cancerthat typically involves weight loss and loss of skeletal muscle mass.Cancer cachexia accounts for more than 20 percent of all cancer-relateddeaths (Murphy K T and Lynch G S: Update on emerging drugs for cancercachexia. Expert Opin Emerg Drugs. 2009 December; 14(4):619-32.).

Thus, in order to improve the treatment and prognosis of cancers whichlead to cancer cachexia, treatment regimens that target both of thesemedical conditions are needed. To date, most of the emerging drugs fortreatments of cancer cachexia are drugs that target cachexia but not thecancer itself (see Murphy K T and Lynch G S: Update on emerging drugsfor cancer cachexia. Expert Opin Emerg Drugs. 2009 December;14(4):619-32.). Only very few drugs are effective against both thecancer and cancer cachexia, and therefore, complex treatment regimensthat combine anti-cancer drugs and anti-cancer cachexia drugs are oftentimes needed. Accordingly, there is still an unmet medical need fordrugs that can be used to effectively treat both cancer and cancercachexia in a broad range of cancers.

Many types of cancer are known to express growth factors, includingfactors such as VEGF, PDGF, TGF-β and GDF-15.

GDF-15, growth and differentiation factor-15, is a divergent member ofthe TGF-β superfamily. It is a protein which is intracellularlyexpressed as a precursor, subsequently processed and eventually becomessecreted from the cell into the environment. Both the active, fullyprocessed (mature) form and the precursor of GDF-15 can be found outsidecells. The precursor covalently binds via its COOH-terminal amino acidsequence to the extracellular matrix (Bauskin A R et al., CancerResearch 2005) and thus resides on the exterior of a cell. The active,fully processed (mature) form of GDF-15 is soluble and is found in bloodsera. Thus, the processed form of GDF-15 may potentially act on anytarget cell within the body that is connected to the blood circulation,provided that the potential target cell expresses a receptor for thesoluble GDF-15 ligand.

During pregnancy, GDF-15 is found under physiological conditions in theplacenta. However, many malignant cancers (especially aggressive braincancers, melanoma, lung cancer, gastrointestinal tumors, colon cancer,pancreatic cancer, prostate cancer and breast cancer (Mimeault M andBatra S K, J. Cell Physiol 2010)) exhibit increased GDF-15 levels in thetumor as well as in blood serum. Likewise, correlations have beendescribed between high GDF-15 expression and chemoresistance (Huang C Yet al., Clin. Cancer Res. 2009) and between high GDF-15 expression andpoor prognosis, respectively (Brown D A et al., Clin. Cancer Res. 2009).

GDF-15 is expressed in gliomas of different WHO grades as assessed byimmunohistochemistry (Roth et al., Clin. Cancer Res. 2010). Further,Roth et al. stably expressed short hairpin. RNA-expressing DNAconstructs targeting endogenous GDF-15 or control constructs in SMA560glioma cells. When using these pre-established stable cell lines, theyobserved that tumor formation in mice bearing GDF-15 knockdown SMA560cells was delayed compared to mice bearing control constructs.

Patent application PCT/EP2013/070127 relates to monoclonal anti-GDF-15antibodies, in particular to an antibody produced by the hybridoma cellline B1-23 deposited with the Deutsche Sammlung für Mikroorganismen undZellkulturen GmbH (DSMZ) under the accession No. DSM ACC3142 under theBudapest treaty. PCT/EP2013/070127 also relates to uses of theanti-GDF-15 antibodies.

Patent applications WO 2005/099746 and WO 2009/021293 relate to ananti-human-GDF-15 antibody (Mab26) capable of antagonizing effects ofhuman GDF-15 on tumor-induced weight loss in vivo in mice: In thesedocuments, immunologically compromised mice were administered with humantumor cells (prostate carcinoma cells DU145) transfected with plasmidsoverexpressing human GDF-15. Tumor cells carrying plasmids lacking aGDF-15 sequence served as a negative control. Those mice expressingxenograft GDF-15 exhibited a tumor-induced weight loss (clinical term:cachexia) and anorexia. A single intraperitoneal administration of 1 mgof Mab26 from WO 2005/099746 resulted in a complete reversal oftumor-induced weight loss. WO 2005/099746 and WO 2009/021293 do notdisclose effects of an anti-human-GDF-15 antibody on tumor growth.Moreover, these documents are silent as to whether anti-human-GDF-15antibodies could lead to an increase in body weight of the treated micecompared to their body weight before the onset of cachexia.

Similarly, Johnen H et al. (Nature Medicine, 2007) reported effects ofan anti-human-GDF-15 monoclonal antibody on cancer-induced anorexia andweight loss but did not observe any effects of the anti-human-GDF-15antibody on the size of the tumor formed by the cancer, even when theantibody was administered at a high dosage of 1 mg, and thus theantibody did not inhibit growth of the cancer.

Accordingly, to date, there was still a need in the art for means toeffectively treat cancer and cancer cachexia, and for means to treatcancer and cancer cachexia in a broader range of cancers.

It is therefore an object of the invention to obtain means that can beused to effectively treat cancer cachexia, and to also effectively treatcancer, and means that can be used to treat cancer cachexia, and to alsoeffectively treat cancer in a broader range of cancers.

In an effort to find means to achieve these objects, the presentinventors have surprisingly found that a monoclonal antibody to humanGDF-15 can be used to treat cancer cachexia and to also treat cancer ofhuman xenograft tumors in mice.

Additionally, an antibody to human GDF-15 in accordance with the presentinvention has an equilibrium dissociation constant of about 790 pM forrecombinant GDF-15 even without additional affinity maturation, which isa higher affinity compared to most known therapeutic antibodies.

Thus, the antibody to human GDF-15 according to the present inventionhas superior properties compared to antibodies known from the art, andis particularly useful for inhibiting cancer growth and cancer cachexia.The antibody of the present invention is therefore useful for treatingcancer and for treating cancer cachexia. Accordingly, the presentinvention was completed.

BRIEF DESCRIPTION OF THE INVENTION

The present invention solves the above-mentioned objects by providingthe monoclonal antibodies, pharmaceutical compositions, kits, uses andthe cell lines capable of producing the monoclonal antibodies describedherein.

In particular, the present inventors surprisingly show that monoclonalantibodies to human GDF-15 and antigen binding portions thereofaccording to the invention are capable of inhibiting cancer cachexiaand/or cancer growth. This was unexpected because those monoclonalantibodies to GDF-15 that were previously known from the art (WO2005/099746, WO 2009/021293 and Johnen H et al., Nature Medicine, 2007)were only known to cause a reversal of cancer-induced weight loss (i.e.a reversal of a secondary symptom induced by the GDF-15 expressed by thecancer), but were shown to fail at inhibiting growth of the cancer.

By showing that the monoclonal antibodies to human GDF-15 according tothe invention can be used to treat cancer-induced weight loss and/orcancer cachexia and treat cancer, the present inventors alsosurprisingly show that human GDF-15 protein can be targeted by theantibodies of the invention in a way that both cancer growth isinhibited and cancer-induced weight loss and cancer cachexia is treated.It is expected that the same mechanisms of cancer growth inhibition andtreatment of cancer-induced weight loss and cancer cachexia areapplicable to a large number of cancers that overexpress human GDF-15including the cancers listed below.

The monoclonal antibodies and antigen-binding portions thereof accordingto the invention are derived from a murine anti-GDF-15 antibody,mAb-B1-23, which was described in PCT/EP2013/070127 and deposited withthe Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (DSMZ)under the accession No. DSM ACC3142 under the Budapest treaty. Theanti-human GDF-15 mAb-B1-23 antibodies according to the invention can begenerated by replacing constant domains of the murine antibody mAb-B1-23with the constant domains of a human IgG1 antibody.

Surprisingly, it was observed that a chimeric and a humanized B1-23antibody according to the invention showed no tendency to aggregate.These antibody properties according to the invention are expected toincrease the bioavailability of these antibodies and to be advantageousfor clinical formulation of these antibodies.

Thus, the present invention relates to a monoclonal antibody capable ofbinding to human GDF-15, or an antigen-binding portion thereof, whereinthe heavy chain variable domain comprises a CDR3 region comprising theamino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least90% identical thereto, and wherein the light chain variable domaincomprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence at least 85% identical thereto wherein theconstant domain of the heavy chain comprises the amino acid sequence ofSEQ ID No: 29, or an amino acid sequence at least 85%, preferably atleast 90%, more preferably at least 95% identical thereto, and whereinthe constant domain of the light chain comprises the amino acid sequenceof SEQ ID No: 32, or an amino acid sequence at least 85%, preferably atleast 90%, more preferably at least 95% identical thereto.

The present invention also relates to a monoclonal antibody capable ofbinding to human GDF-15, or an antigen-binding portion thereof, whereinthe heavy chain variable domain comprises a CDR3 region comprising theamino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least90% identical thereto, and wherein the light chain variable domaincomprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence at least 85% identical thereto, for use in amethod for treating cancer cachexia in a mammal. The method comprisesadministering the antibody or antigen-binding portion thereof to saidmammal. Additionally, the present invention relates to a correspondingmethod for treatment.

Further, the invention also relates to a monoclonal antibody capable ofbinding to human GDF-15, or an antigen-binding portion thereof, whereinthe binding is binding to a conformational or discontinuous epitope onhuman GDF-15 comprised by the amino acid sequences of SEQ ID No: 25 andSEQ ID No: 26, wherein the constant domain of the heavy chain comprisesthe amino acid sequence of SEQ ID No: 29, or an amino acid sequence atleast 85%, preferably at least 90%, more preferably at least 95%identical thereto, and wherein the constant domain of the light chaincomprises the amino acid sequence of SEQ ID No: 32, or an amino acidsequence at least 85%, preferably at least 90%, more preferably at least95% identical thereto.

Further, the invention also relates to a monoclonal antibody capable ofbinding to human GDF-15, or an antigen-binding portion thereof, whereinthe binding is binding to a conformational or discontinuous epitope onhuman GDF-15 comprised by the amino acid sequences of SEQ ID No: 25 andSEQ ID No: 26, for use in a method for treating cancer cachexia in amammal. The method comprises administering the antibody orantigen-binding portion thereof to said mammal. Additionally, thepresent invention relates to a corresponding method for treatment.

The invention also relates to a pharmaceutical composition comprisingthe antibody or antigen-binding portion thereof according to theinvention.

The invention also relates to an antibody or antigen-binding portionthereof according to the invention for use in medicine.

Further, the invention relates to an antibody or antigen-binding portionthereof or a pharmaceutical composition according to the invention foruse in a method for treating cancer in a mammal. The method comprisesadministering the antibody or antigen-binding portion thereof or thepharmaceutical composition to said mammal.

Further, the invention relates to an antibody or antigen-binding portionthereof or a pharmaceutical composition according to the invention foruse in a method for treating cancer cachexia in a mammal. The methodcomprises administering the antibody or antigen-binding portion thereofor the pharmaceutical composition to said mammal.

Additionally, the invention relates to a kit comprising thepharmaceutical composition according to the invention.

The invention also relates to an expression vector comprising anucleotide sequence encoding the antibody or antigen-binding portionthereof according to the invention.

Further, the invention relates to a cell line capable of producing anantibody or antigen-binding portion thereof according to the invention.

Thus, by providing monoclonal antibodies to human GDF-15, the presentinvention provides means for the treatment of cancer cachexia and acancer growth inhibitor that meets the above-defined needs in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: NKG2D Expression on NK Cells after Treatment with or withoutGDF-15. The cell surface expression of NKG2D was determined on NK cellsafter treatment with the indicated cytokines in the presence or absenceof the anti-GDF-15 antibody mAb B1-23. The figure displays specificfluorescence intensities determined by flow cytometry, quantifiedrelative to an unspecific control antibody.

FIG. 2: Akt Phosphorylation in the Ovarian Carcinoma Cell Line SK-OV-3.In order to quantify the Western Blot for the ovarian carcinoma cellline SK-OV-3, the ratio of phosphorylated Akt to the total amount of Aktwas calculated and normalized to the untreated control.

FIG. 3: JNK1/2 Phosphorylation in Immune Cells. In order to quantify theWestern Blot, the ratio of phosphorylated JNK1/2 to the total amount ofJNK was calculated and normalized to the untreated control.

FIG. 4:

An anti-tumor effect of murine B1-23 in vivo. Balb/c^(nu/nu) nude micewere used in a xenograft setting with the melanoma cell line UACC-257.The tumor size of the animal cohort treated with B1-23 (open squares)was significantly decreased, compared to the PBS control group (filledsolid circles). Significance was defined as p<0.05 as assessed byWilcoxon's log-rank test.

FIG. 5: Treatment of cancer cachexia with anti-GDF-15 antibodies. Thefigure shows a comparison of the mean body weight of all treatedBalb/c^(nu/nu) nude mice, which were inoculated with UACC-257 cells. Thechanges of the body weight are depicted in percent as compared to thestarting body weight on day 0, for a period of 38 days.

FIG. 6: Coomassie stain of antibodies used in the study No. 140123.

FIG. 7: Improved solubility of the chimeric and the humanized antibodyat physiological pH.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise defined below, the terms used in the present inventionshall be understood in accordance with their common meaning known to theperson skilled in the art.

The term “antibody” as used herein refers to any functional antibodythat is capable of specific binding to the antigen of interest, asgenerally outlined in chapter 7 of Paul, W. E. (Ed.).: FundamentalImmunology 2nd Ed. Raven Press, Ltd., New York 1989, which isincorporated herein by reference. Without particular limitation, theterm “antibody” encompasses antibodies from any appropriate sourcespecies, including chicken and mammalian such as mouse, goat, non-humanprimate and human. Preferably, the antibody is a humanized antibody. Theantibody is preferably a monoclonal antibody which can be prepared bymethods well-known in the art. The term “antibody” encompasses an IgG-1,-2, -3, or -4, IgE, IgA, IgM, or IgD isotype antibody. The term“antibody” encompasses monomeric antibodies (such as IgD, IgE, IgG) oroligomeric antibodies (such as IgA or IgM). The term “antibody” alsoencompasses—without particular limitations—isolated antibodies andmodified antibodies such as genetically engineered antibodies, e.g.chimeric antibodies.

The nomenclature of the domains of antibodies follows the terms as knownin the art. Each monomer of an antibody comprises two heavy chains andtwo light chains, as generally known in the art. Of these, each heavyand light chain comprises a variable domain (termed V_(H) for the heavychain and V_(L) for the light chain) which is important for antigenbinding. These heavy and light chain variable domains comprise (in anN-terminal to C-terminal order) the regions FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4 (FR, framework region; CDR, complementarity determiningregion which is also known as hypervariable region). The identificationand assignment of the above-mentioned antibody regions within theantibody sequence is generally in accordance with Kabat et al.(Sequences of proteins of immunological interest, U.S. Dept. of Healthand Human Services, Public Health Service, National Institutes ofHealth, Bethesda, Md. 1983), or Chothia et al. (Conformations ofimmunoglobulin hypervariable regions. Nature. 1989 Dec. 21-28;342(6252):877-83.), or may be performed by using the IMGT/V-QUESTsoftware described in Giudicelli et al. (IMGT/V-QUEST, an integratedsoftware program for immunoglobulin and T cell receptor V-J and V-D-Jrearrangement analysis. Nucleic Acids Res. 2004 Jul. 1; 32(Web Serverissue):W435-40.), which is incorporated herein by reference. Preferably,the antibody regions indicated above are identified and assigned byusing the IMGT/V-QUEST software.

A “monoclonal antibody” is an antibody from an essentially homogenouspopulation of antibodies, wherein the antibodies are substantiallyidentical in sequence (i.e. identical except for minor fraction ofantibodies containing naturally occurring sequence modifications such asamino acid modifications at their N- and C-termini). Unlike polyclonalantibodies which contain a mixture of different antibodies directed tonumerous epitopes, monoclonal antibodies are directed to the sameepitope and are therefore highly specific. The term “monoclonalantibody” includes (but is not limited to) antibodies which are obtainedfrom a monoclonal cell population derived from a single cell clone, asfor instance the antibodies generated by the hybridoma method describedin Köhler and Milstein (Nature, 1975 Aug. 7; 256(5517):495-7) or Harlowand Lane (“Antibodies: A Laboratory Manual” Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. 1988). A monoclonal antibodymay also be obtained from other suitable methods, including phagedisplay techniques such as those described in Clackson et al. (Nature.1991 Aug. 15; 352(6336):624-8) or Marks et al. (J Mol Biol. 1991 Dec. 5;222(3):581-97). A monoclonal antibody may be an antibody that has beenoptimized for antigen-binding properties such as decreased Kd values,optimized association and dissociation kinetics by methods known in theart. For instance, Kd values may be optimized by display methodsincluding phage display, resulting in affinity-matured monoclonalantibodies. The term “monoclonal antibody” is not limited to antibodysequences from particular species of origin or from one single speciesof origin. Thus, the meaning of the term “monoclonal antibody”encompasses chimeric monoclonal antibodies such as humanized monoclonalantibodies.

“Humanized antibodies” are antibodies which contain human sequences anda minor portion of non-human sequences which confer binding specificityto an antigen of interest (e.g. human GDF-15). Typically, humanizedantibodies are generated by replacing hypervariable region sequencesfrom a human acceptor antibody by hypervariable region sequences from anon-human donor antibody (e.g. a mouse, rabbit, rat donor antibody) thatbinds to an antigen of interest (e.g. human GDF-15). In some cases,framework region sequences of the acceptor antibody may also be replacedby the corresponding sequences of the donor antibody. In addition to thesequences derived from the donor and acceptor antibodies, a “humanizedantibody” may either contain other (additional or substitute) residuesor sequences or not. Such other residues or sequences may serve tofurther improve antibody properties such as binding properties (e.g. todecrease Kd values) and/or immunogenic properties (e.g. to decreaseantigenicity in humans). Non-limiting examples for methods to generatehumanized antibodies are known in the art, e.g. from Riechmann et al.(Nature. 1988 Mar. 24; 332(6162):323-7) or Jones et al. (Nature. 1986May 29-Jun. 4; 321(6069):522-5).

The term “human antibody” relates to an antibody containing humanvariable and constant domain sequences. This definition encompassesantibodies having human sequences bearing single amino acidsubstitutions or modifications which may serve to further improveantibody properties such as binding properties (e.g. to decrease Kdvalues) and/or immunogenic properties (e.g. to decrease antigenicity inhumans). The term “human antibody” excludes humanized antibodies where aportion of non-human sequences confers binding specificity to an antigenof interest.

An “antigen-binding portion” of an antibody as used herein refers to aportion of an antibody that retains the capability of the antibody tospecifically bind to the antigen (e.g. GDF-15), i.e. the“antigen-binding portion” is capable of competing with the antibody forspecific binding to the antigen. The “antigen-binding portion” maycontain one or more fragments of the antibody. Without particularlimitation, it can be produced by any suitable method known in the art,including recombinant DNA methods and preparation by chemical orenzymatic fragmentation of antibodies. Antigen-binding portions may beFab fragments, F(ab′) fragments, F(ab′)₂ fragments, single chainantibodies (scFv), single-domain antibodies, diabodies or any otherportion(s) of the antibody that allow(s) to retain binding to theantigen.

An “antibody” (e.g. a monoclonal antibody) or an “antigen-bindingportion” may have been derivatized or be linked to a different molecule.For example, molecules that may be linked to the antibody are otherproteins (e.g. other antibodies), a molecular label (e.g. a fluorescent,luminescent, colored or radioactive molecule), a pharmaceutical and/or atoxic agent. The antibody or antigen-binding portion may be linkeddirectly (e.g. in form of a fusion between two proteins), or via alinker molecule (e.g. any suitable type of chemical linker known in theart).

As used herein, the terms “binding” or “bind” refer to specific bindingto the antigen of interest (e.g. human GDF-15). Preferably, the Kd valueis less than 100 nM, more preferably less than 50 nM, still morepreferably less than nM, still more preferably less than 5 nM and mostpreferably less than 2 nM.

The term “epitope” as used herein refers to a small portion of anantigen that forms the binding site for an antibody.

In the context of the present invention, binding or competitive bindingof antibodies or their antigen-binding portions to the antigen ofinterest (e.g. human GDF-15) is measured by using surface plasmonresonance measurements as a reference standard assay, as describedbelow.

The terms “K_(D)” or “K_(D) value” relate to the equilibriumdissociation constant as known in the art. In the context of the presentinvention, these terms relate to the equilibrium dissociation constantof an antibody with respect to a particular antigen of interest (e.g.human GDF-15). The equilibrium dissociation constant is a measure of thepropensity of a complex (e.g. an antigen-antibody complex) to reversiblydissociate into its components (e.g. the antigen and the antibody). Forthe antibodies according to the invention, K_(D) values (such as thosefor the antigen human GDF-15) are generally determined by using surfaceplasmon resonance measurements as described below.

The term “cancer growth” as used herein relates to any measureablegrowth of the cancer. For cancers forming solid tumors, “cancer growth”relates to a measurable increase in tumor volume over time. If thecancer has formed only a single tumor, “cancer growth” relates only tothe increase in volume of the single tumor. If the cancer has formedmultiple tumors such as metastases, “cancer growth” relates to theincrease in volume of all measurable tumors. For solid tumors, the tumorvolume can be measured by any method known in the art, includingmagnetic resonance imaging and computed tomography (CT scan).

For leukemias which are characterized by the presence of cancerous cellsof the blood system in blood, “cancer growth” relates to a measurableincrease in the number of cancer cells per blood volume. In order tocarry out such measurements, cancer cells can be identified from bloodsamples by using any method known in the art, including cell morphologymeasurements, or staining of tumor cell marker proteins such as tumormarker cell surface proteins, e.g. by staining with specific antibodies,and the cancer cells can be counted.

Terms such as “inhibiting cancer growth” as used herein refer to ameasurable inhibition of cancer growth in patient treated with theantibody. Preferably, the inhibition is statistically significant.Inhibition of cancer growth may be assessed by comparing cancer growthin a group of patients treated in accordance with the present inventionto a control group of untreated patients, or by comparing a group ofpatients that receive a standard cancer treatment of the art plus atreatment according to the invention with a control group of patientsthat only receive a standard cancer treatment of the art. Such studiesfor assessing the inhibition of cancer growth are designed in accordancewith accepted standards for clinical studies, e.g. double-blinded,randomized studies with sufficient statistical power. The term“inhibiting cancer growth” includes an inhibition of cancer growth wherethe cancer growth is inhibited partially (i.e. where the cancer growthin the patient is delayed compared to the control group of patients), aninhibition where the cancer growth is inhibited completely (i.e. wherethe cancer growth in the patient is stopped), and an inhibition wherecancer growth is reversed (i.e. the cancer shrinks).

An “isolated antibody” as used herein is an antibody that has beenidentified and separated from the majority of components (by weight) ofits source environment, e.g. from the components of a hybridoma cellculture or a different cell culture that was used for its production(e.g. producer cells such as CHO cells that recombinantly express theantibody). The separation is performed such that it sufficiently removescomponents that may otherwise interfere with the suitability of theantibody for the desired applications (e.g. with a therapeutic use ofthe anti-human GDF-15 antibody according to the invention). Methods forpreparing isolated antibodies are known in the art and include Protein Achromatography, anion exchange chromatography, cation exchangechromatography, virus retentive filtration and ultrafiltration.Preferably, the isolated antibody preparation is at least 70% pure(w/w), more preferably at least 80% pure (w/w), still more preferably atleast 90% pure (w/w), still more preferably at least 95% pure (w/w), andmost preferably at least 99% pure (w/w), as measured by using the Lowryprotein assay.

A “diabody” as used herein is a small bivalent antigen-binding antibodyportion which comprises a heavy chain variable domain linked to a lightchain variable domain on the same polypeptide chain linked by a peptidelinker that is too short to allow pairing between the two domains on thesame chain. This results in pairing with the complementary domains ofanother chain and in the assembly of a dimeric molecule with two antigenbinding sites. Diabodies may be bivalent and monospecific (such asdiabodies with two antigen binding sites for human GDF-15), or may bebivalent and bispecific (e.g. diabodies with two antigen binding sites,one being a binding site for human GDF-15, and the other one being abinding site for a different antigen). A detailed description ofdiabodies can be found in Holliger P et al. (““Diabodies”: smallbivalent and bispecific antibody fragments.” Proc Natl Acad Sci USA.1993 Jul. 15; 90(14):6444-8.).

A “single-domain antibody” (which is also referred to as “Nanobody™”) asused herein is an antibody fragment consisting of a single monomericvariable antibody domain. Structures of and methods for producingsingle-domain antibodies are known from the art, e.g. from Holt L J etal. (“Domain antibodies: proteins for therapy.” Trends Biotechnol. 2003November; 21(11):484-90.), Saerens D et al. (“Single-domain antibodiesas building blocks for novel therapeutics.” Curr Opin Pharmacol. 2008October; 8(5):600-8. Epub 2008 Aug. 22.), and Arbabi Ghahroudi M et al.(“Selection and identification of single domain antibody fragments fromcamel heavy-chain antibodies.” FEBS Lett. 1997 Sep. 15; 414(3):521-6.).

The term “higher” as used herein means that a value (e.g. a GDF-15level) in a patient sample is higher than a value in a correspondingcontrol sample or group of control samples. Preferably, the differenceis statistically significant.

The term “elevated GDF-15 levels” as used herein means that the humanpatient has higher GDF-15 levels in blood serum before administration ofthe antibody or antigen-binding portion thereof or the pharmaceuticalcomposition according to the invention, when compared to median GDF-15levels in blood sera of healthy human control individuals as areference.

A preferred median reference for GDF-15 level in blood sera of healthyhuman control individuals is <0.8 ng/ml. The expected range is between0.2 ng/ml and 1.2 ng/ml in healthy human controls (Reference: Tanno T etal.: “Growth differentiation factor 15 in erythroid health and disease.”Curr Opin Hematol. 2010 May; 17(3): 184-190.).

Preferably, the levels are 1.2-fold higher, more preferably 1.5-foldhigher, still more preferably 2-fold higher and most preferably 5-foldhigher.

The term “prior to administration” as used herein means the period oftime immediately before administration of the antibody, fragment thereofor the pharmaceutical composition according to the invention.Preferably, the term “prior to administration” means a period of 30 daysimmediately before administration; most preferably a period of one weekimmediately before administration.

The terms “significant”, “significantly”, etc. as used herein refer to astatistically significant difference between values.

The terms “cancer” and “cancer cell” is used herein in accordance withtheir common meaning in the art (see for instance Weinberg R. et al.:The Biology of Cancer. Garland Science: New York 2006. 850p.).

The term “cancer-induced weight loss” is used herein in accordance withits common meaning in the art. Cancer-induced weight loss is frequentlyseen as an adverse effect in individuals having cancer (see, forinstance Fearon K. et al.: Definition and classification of cancercachexia: an international consensus. Lancet Oncol. 2011 May;12(5):489-95.; Tisdale M J.: Mechanisms of cancer cachexia. Physiol Rev.2009 April; 89(2):381-410.). The term “cancer-induced weight loss”relates to the body weight loss induced by the cancer. Additional bodyweight loss in addition to the cancer-induced weight loss—e.g. bodyweight loss induced by cancer treatments such as surgery, chemotherapyand radiotherapy can also occur in individuals having cancer. It isunderstood that the meaning of the term “cancer-induced weight loss”does not include this additional body weight loss. However, this doesnot exclude the possibility that the antibodies of the presentinvention—in addition to their effects on cancer-induced weight loss andon cancer growth—may have beneficial effects against such additionalbody weight loss, e.g. by reverting or partly reverting such additionalweight loss, or by preventing or partly preventing such additional bodyweight loss.

Body weight can easily be measured by weighing, and body weight istypically expressed in units of mass such as kg.

The term “cancer cachexia” is used herein in accordance with its commonmeaning in the art (see, for instance Fearon K. et al.: Definition andclassification of cancer cachexia: an international consensus. LancetOncol. 2011 May; 12(5):489-95.; Tisdale M J.: Mechanisms of cancercachexia. Physiol Rev. 2009 April; 89(2):381-410.). The most commonsymptom of cancer cachexia is cancer-induced weight loss. Thus,according to one definition, cancer cachexia is characterized by anongoing loss of skeletal muscle mass (with or without loss of fat mass)that cannot be fully reversed by conventional nutrition. In humanpatients, cancer cachexia can be defined by a weight loss of more than5% during the past 6 months, or by a body mass index of less than 20g/m² and any degree of ongoing weight loss that is higher than 2%, or bysarcopenia (i.e. degenerative loss of muscle mass) and an ongoing weightloss that is higher than 2% (see, Fearon K. et al.: Definition andclassification of cancer cachexia: an international consensus. LancetOncol. 2011 May; 12(5):489-95.). A further symptom of cancer cachexiacan be a depletion of adipose tissue.

With respect to cancer cachexia, a “treatment” according to the presentinvention may be a treatment for preventing and/or a treatment forinhibiting or reverting cancer cachexia. Typically, a treatment forpreventing cancer cachexia is a treatment that is given prophylacticallyat a stage of the cancer disease where no cancer cachexia has yetoccurred. A treatment for inhibiting cancer cachexia is typically atreatment that is given at a stage of the cancer disease where somecancer cachexia has occurred, in order to inhibit a further progressionof the cancer cachexia. A treatment for reverting cancer cachexia istypically a treatment that is started at a stage of the cancer wheresome cancer cachexia has occurred, and which reverts the cancercachexia. The effect of the treatment can be a partial effect, i.e. apartial prevention, a partial inhibition or a partial reversion ofcancer cachexia, or a complete effect, i.e. a complete prevention, acomplete inhibition or a complete reversion of cancer cachexia.Preferably, according to the present invention, the effect of thetreatment is a complete prevention, a complete inhibition or a completereversion of cancer cachexia. More preferably, the effect of thetreatment according to the present invention is a complete prevention ora complete reversion of cancer cachexia.

As used herein, the term “complete(ly)” in connection with a treatmentof cancer cachexia according to the invention means that in case of atreatment for preventing, no cancer cachexia occurs in the treatedindividual during and/or following the treatment. In case of a treatmentfor inhibiting cancer cachexia, the term “complete(ly)” means that nofurther progression of the cancer cachexia occurs in the treatedindividual during and/or following the treatment. In case of a treatmentfor reverting cancer cachexia, the term “complete(ly)” means that duringor following the treatment, the cancer cachexia is completely revertedsuch that no cancer cachexia is present in the treated individual.

With respect to these effects of the treatment according to theinvention, the term “no cancer cachexia” means that by using standardmethods for measurements and for diagnosis known in the art, no cancercachexia is detectable. Likewise, the term “no further progression ofthe cancer cachexia” means that by using standard methods formeasurements and for diagnosis known in the art, no further progressionof cancer cachexia is detectable. The methods known in the art andreferred to herein are for instance described in Fearon K C.: Cancercachexia: developing multimodal therapy for a multidimensional problem.Eur J Cancer. 2008 May; 44(8):1124-32; Fearon K. et al.: Definition andclassification of cancer cachexia: an international consensus. LancetOncol. 2011 May; 12(5):489-95.; or Tisdale M J.: Mechanisms of cancercachexia. Physiol Rev. 2009 April; 89(2):381-410.

In addition to completely preventing or completely reverting cancercachexia, the treatment methods and products for use in these methodsaccording to the invention may increase the body weight of the treatedmammal compared to its body weight before the onset of cancer cachexia.As used herein, the term “before the onset of cancer cachexia” means apoint in time during the course of the cancer disease, after whichcancer cachexia becomes measurable by the methods known in the art suchas the methods referred to above.

Preferably, the above-defined effects of the cancer cachexia treatmentaccording to the invention are statistically significant when assessedagainst a suitable control group whereas individual patients who aretreated would not show significant cachexia.

In accordance with the present invention, each occurrence of the term“comprising” may optionally be substituted with the term “consistingof”.

Methods and Techniques

Generally, unless otherwise defined herein, the methods used in thepresent invention (e.g. cloning methods or methods relating toantibodies) are performed in accordance with procedures known in theart, e.g. the procedures described in Sambrook et al. (“MolecularCloning: A Laboratory Manual.”, 2^(nd) Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. 1989), Ausubel et al.(“Current Protocols in Molecular Biology.” Greene Publishing Associatesand Wiley Interscience; New York 1992), and Harlow and Lane(“Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. 1988), all of which are incorporated herein byreference.

Molecular weight is measured by methods known in the art such as massspectrometry. It is expressed in Dalton (Da) or Kilodalton (kDa).

Binding of monoclonal anti-human-GDF-15 antibodies according to theinvention is generally assessed by employing surface plasmon resonancemeasurements using a Bio-Rad® ProteOn™ XPR36 system and Bio-Rad® GLCsensor chips as described for murine anti-human GDF-15 mAb-B1-23 inExample 1.

Sequence alignments of sequences according to the invention areperformed by using the BLAST algorithm (see Altschul et al. (1990)“Basic local alignment search tool.” Journal of Molecular Biology 215.p. 403-410.; Altschul et al.; (1997) Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucleic Acids Res.25:3389-3402.). Preferably, the following parameters are used: Maxtarget sequences 10; Word size 3; BLOSUM 62 matrix; gap costs: existence11, extension 1; conditional compositional score matrix adjustment.Thus, when used in connection with sequences, terms such as “identity”or “identical” refer to the identity value obtained by using the BLASTalgorithm.

Monoclonal antibodies according to the invention can be produced by anymethod known in the art, including but not limited to the methodsreferred to in Siegel D L (“Recombinant monoclonal antibody technology.”Transfus Clin Biol. 2002 January; 9(1):15-22.). In a preferredembodiment, an antibody according to the invention is produced by thehybridoma cell line B1-23 deposited with the Deutsche Sammlung fürMikroorganismen und Zellkulturen GmbH (DSMZ) under the accession No. DSMACC3142 under the Budapest treaty. The deposit was filed on Sep. 29,2011.

Cell proliferation can be measured by suitable methods known in the art,including (but not limited to) visual microscopy, metabolic assays suchas those which measure mitochondrial redox potential (e.g. MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay;Resazurin staining which is also known as Alamar Blue® assay), stainingof known endogenous proliferation biomarkers (e.g. Ki-67), and methodsmeasuring cellular DNA synthesis (e.g. BrdU and [³H]-Thymidineincorporation assays).

Immunosuppression can be measured by suitable methods known in the art,including (but not limited to) immune cell proliferation, cytokinesecretion, intracellular cytokine staining by flow cytometry, cytokinemeasurement by qRT-PCR, redirected target cell lysis, furthercytotoxicity or degranulation assays, downregulation of activatingimmune cell receptors (like NKG2D), upregulation of inhibitory immunecell receptors, immunological synapse formation, immune cellinfiltration. For the term immunosuppression to apply, an effect shallbe measurable in at least one of these or in any other suitable assay.The lack of effect in a specific test does not imply a general absenceof immunosuppression.

Human GDF-15 levels can be measured by any method known in the art,including measurements of GDF-15 mRNA levels by methods including (butnot limited to) quantitative real-time PCR (qRT-PCR) for human GDF-15mRNA using primers specific to human GDF-15, mRNA in situ hybridizationwith probes specific to human GDF-15, mRNA deep sequencing methods; andincluding measurements of GDF-15 protein levels by methods including(but not limited to) mass spectrometry for proteins or peptides derivedfrom human GDF-15, Western Blotting using antibodies specific to humanGDF-15, flow cytometry using antibodies specific to human GDF-15, striptests using antibodies specific to human GDF-15, or immunocytochemistryusing antibodies specific to human GDF-15. For such methods usingantibodies specific to human GDF-15, the anti-human GDF-15 antibodies ofthe present invention are preferred, and the antibody of the inventionproduced by the hybridoma cell line B1-23 deposited with the DeutscheSammlung für Mikroorganismen und Zellkulturen GmbH (DSMZ) under theaccession No. DSM ACC3142 is most preferred.

EMBODIMENTS OF THE INVENTION

As described above, the inventors show that human GDF-15 protein can betargeted by an antibody in accordance with the invention in a way thatcancer cachexia and cancer-induced weight loss can be treated and thatalso cancer growth is inhibited.

When taking into account the present invention, it becomes clear thatthe anti-GDF-15 antibodies known from WO 2005/099746, WO 2009/021293 andJohnen H et al., Nature Medicine, 2007 only inhibit one of the effectsof human GDF-15 (i.e. cancer-induced weight loss), but fail to inhibitother effects of human GDF-15 such as those related to cancer growth. Inview of the present invention, one possible explanation for this failureis that the antibodies known from the above documents may only interferewith transport of human GDF-15 across the blood-brain barrier (byforming a large complex that cannot be transported across theblood-brain barrier) but are incapable of binding human GDF-15 in a waythat renders it generally unable to interact with its receptor (e.g. areceptor residing on cells outside the brain). Furthermore, anddifferent from the antibodies of the present invention, the anti-GDF-15antibodies known from WO 2005/099746, WO 2009/021293 and Johnen H etal., Nature Medicine, 2007 did not lead to a detectable increase in thebody weight of the mammals compared to its body weight before the onsetof cancer cachexia.

Accordingly, the effects of the antibodies for use according to theinvention are unexpected in view of the art.

The following properties of the antibodies of the present invention areexpected to contribute to their capability of inhibiting the effects ofhuman GDF-15 more completely, including the treatment of cachexia andthe inhibition of cancer growth:

Broad Binding Specificity to Forms of Human GDF-15

The antibodies of the present invention are capable of binding to maturerecombinant human GDF-15 (represented by SEQ ID No: 8) and are thereforecapable of binding to active, fully processed (mature) human GDF-15.

Additionally, by performing staining experiments with the murinemAb-B1-23 antibody according to the invention on human cells, theinventors show that the mAb-B1-23 antibody according to the invention iscapable of binding to the human GDF-15 precursor on human cells.

Thus, it is expected that binding and effects of the antibodiesaccording to the present invention, in particular the inhibition ofcancer growth, are not generally limited to effects on a particular formof human GDF-15.

As to the effects of human GDF-15 on cancer cachexia, these effects maybe caused a subset of forms human GDF-15, for instance to soluble formshuman GDF-15, which are capable of passing the blood-brain barrier. Asexemplified in the Examples of the present invention, all of the testedanti-GDF-15 antibodies according to the invention can be used to treatcancer-induced cachexia. Thus, the antibodies according to the presentinvention can interfere with the forms of human GDF-15 which areresponsible for cancer cachexia.

High Binding Affinity

The antibodies and antigen binding portions thereof according to theinvention have high binding affinity, as demonstrated by the mAb-B1-23antibody according to the invention which has an equilibriumdissociation constant of about 790 pM for recombinant human GDF-15.Notably, such affinity values are superior to most of the existingtherapeutic antibodies, e.g. to the therapeutic antibody Rituximab whichhas an equilibrium dissociation constant of about 8 nM.

High binding affinity will ensure that the antibody to human GDF-15according to the invention stably binds to human GDF-15, such thateffects of human GDF-15 including effects on cancer growth areeffectively inhibited. Likewise, stable binding of the antibodiesaccording to the invention is expected to ensure that forms of humanGDF-15 which cause cancer cachexia cannot carry out their pathologicalfunction. This may for instance be due to an antibody-dependentsequestration of these forms of human GDF-15 from their possible site ofaction in the brain. Such binding and sequestration may for instancetake place at the site of the cancer, or the antibodies according to theinvention may interfere with the transport of human-GDF-15 across theblood brain-barrier.

Binding to a Discontinuous or Conformational Epitope

The antibodies and antigen binding portions thereof according to theinvention bind to a discontinuous or conformational epitope, asdemonstrated below for a murine mAb-B1-23 antibody according to theinvention.

Binding of antibodies and antigen binding portions thereof according tothe invention to a discontinuous or conformational GDF-15 epitope mayhelp to keep human GDF-15 in a specific conformation. Thisconformation-specificity may be advantageous to keep GDF-15 in a formthat cannot be released from the tumor, or that cannot cross the bloodbrain-barrier and cause cancer cachexia at a possible site of action inthe brain. Additionally, such binding to a discontinuous orconformational GDF-15 epitope may contribute to the effective inhibitionof effects of human GDF-15 including effects on cancer growth, e.g. bykeeping GDF-15 in a conformation that cannot functionally interact withits receptor.

Thus, the invention relates to the following embodiments:

A) ANTIBODIES, VECTORS AND CELL LINES

Concretely, the invention relates to a monoclonal antibody capable ofbinding to human GDF-15, or an antigen-binding portion thereof, whereinthe heavy chain variable domain comprises a CDR3 region comprising theamino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least90% identical thereto, and wherein the light chain variable domaincomprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence at least 85% identical thereto, wherein theconstant domain of the heavy chain comprises the amino acid sequence ofSEQ ID No: 29, or an amino acid sequence at least 85%, preferably atleast 90%, more preferably at least 95% identical thereto, and whereinthe constant domain of the light chain comprises the amino acid sequenceof SEQ ID No: 32, or an amino acid sequence at least 85%, preferably atleast 90%, more preferably at least 95% identical thereto.

In a preferred aspect of this embodiment, the constant domain of theheavy chain comprises the amino acid sequence of SEQ ID No: 29, or anamino acid sequence at least 98%, preferably at least 99% identicalthereto, and the constant domain of the light chain comprises the aminoacid sequence of SEQ ID No: 32, or an amino acid sequence at least 98%,preferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises an amino acid sequence at least 85% identicalto the amino acid sequence of SEQ ID No: 29, and the constant domain ofthe light chain comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises an amino acid sequence at least 90% identicalto the amino acid sequence of SEQ ID No: 29, and the constant domain ofthe light chain comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises an amino acid sequence at least 95% identicalto the amino acid sequence of SEQ ID No: 29, and the constant domain ofthe light chain comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises an amino acid sequence at least 98% identicalto the amino acid sequence of SEQ ID No: 29, and the constant domain ofthe light chain comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises an amino acid sequence at least 99% identicalto the amino acid sequence of SEQ ID No: 29, and the constant domain ofthe light chain comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises the amino acid sequence of SEQ ID No: 29, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto, and the constant domain ofthe light chain comprises an amino acid sequence at least 85% identicalto the amino acid sequence of SEQ ID No: 32.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises the amino acid sequence of SEQ ID No: 29, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto, and the constant domain ofthe light chain comprises an amino acid sequence at least 90% identicalto the amino acid sequence of SEQ ID No: 32.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises the amino acid sequence of SEQ ID No: 29, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto, and the constant domain ofthe light chain comprises an amino acid sequence at least 95% identicalto the amino acid sequence of SEQ ID No: 32.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises the amino acid sequence of SEQ ID No: 29, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto, and the constant domain ofthe light chain comprises an amino acid sequence at least 98% identicalto the amino acid sequence of SEQ ID No: 32.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises the amino acid sequence of SEQ ID No: 29, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto, and the constant domain ofthe light chain comprises an amino acid sequence at least 99% identicalto the amino acid sequence of SEQ ID No: 32.

In this embodiment, most preferably, the constant domain of the heavychain comprises the amino acid sequence of SEQ ID No: 29, and theconstant domain of the light chain comprises the amino acid sequence ofSEQ ID No: 32.

In an alternative embodiment, the invention relates to a monoclonalantibody capable of binding to human GDF-15, or an antigen-bindingportion thereof, wherein the heavy chain variable domain comprises aCDR3 region comprising the amino acid sequence of SEQ ID NO: 5 or anamino acid sequence that differs by not more than one amino acid fromthe amino acid sequence of SEQ ID NO: 5, and wherein the light chainvariable domain comprises a CDR3 region comprising the amino acidsequence of SEQ ID NO: 7 or an amino acid sequence or an amino acidsequence that differs by not more than one amino acid from the aminoacid sequence of SEQ ID NO: 7, wherein the constant domain of the heavychain comprises the amino acid sequence of SEQ ID No: 29, or an aminoacid sequence at least 85%, preferably at least 90%, more preferably atleast 95% identical thereto, and wherein the constant domain of thelight chain comprises the amino acid sequence of SEQ ID No: 32, or anamino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95% identical thereto.

In a preferred aspect of this embodiment, the constant domain of theheavy chain comprises the amino acid sequence of SEQ ID No: 29, or anamino acid sequence at least 98%, preferably at least 99% identicalthereto, and the constant domain of the light chain comprises the aminoacid sequence of SEQ ID No: 32, or an amino acid sequence at least 98%,preferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises an amino acid sequence at least 85% identicalto the amino acid sequence of SEQ ID No: 29, and the constant domain ofthe light chain comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises an amino acid sequence at least 90% identicalto the amino acid sequence of SEQ ID No: 29, and the constant domain ofthe light chain comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises an amino acid sequence at least 95% identicalto the amino acid sequence of SEQ ID No: 29, and the constant domain ofthe light chain comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises an amino acid sequence at least 98% identicalto the amino acid sequence of SEQ ID No: 29, and the constant domain ofthe light chain comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises an amino acid sequence at least 99% identicalto the amino acid sequence of SEQ ID No: 29, and the constant domain ofthe light chain comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises the amino acid sequence of SEQ ID No: 29, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto, and the constant domain ofthe light chain comprises an amino acid sequence at least 85% identicalto the amino acid sequence of SEQ ID No: 32.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises the amino acid sequence of SEQ ID No: 29, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto, and the constant domain ofthe light chain comprises an amino acid sequence at least 90% identicalto the amino acid sequence of SEQ ID No: 32.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises the amino acid sequence of SEQ ID No: 29, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto, and the constant domain ofthe light chain comprises an amino acid sequence at least 95% identicalto the amino acid sequence of SEQ ID No: 32.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises the amino acid sequence of SEQ ID No: 29, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto, and the constant domain ofthe light chain comprises an amino acid sequence at least 98% identicalto the amino acid sequence of SEQ ID No: 32.

In another preferred aspect of this embodiment, the constant domain ofthe heavy chain comprises the amino acid sequence of SEQ ID No: 29, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%, and mostpreferably at least 99% identical thereto, and the constant domain ofthe light chain comprises an amino acid sequence at least 99% identicalto the amino acid sequence of SEQ ID No: 32.

In this embodiment, most preferably, the constant domain of the heavychain comprises the amino acid sequence of SEQ ID No: 29, and theconstant domain of the light chain comprises the amino acid sequence ofSEQ ID No: 32.

Further, a monoclonal antibody capable of binding to human GDF-15, or anantigen-binding portion thereof is provided, wherein the heavy chainvariable domain comprises a CDR3 region comprising the amino acidsequence of SEQ ID NO: 5 or an amino acid sequence at least 90%identical thereto, and wherein the light chain variable domain comprisesa CDR3 region comprising the amino acid sequence of SEQ ID NO: 7 or anamino acid sequence at least 85% identical thereto. Preferably, theconstant domain of the heavy chain of this monoclonal antibody orantigen-binding portion thereof comprises the amino acid sequence of SEQID No: 29, or an amino acid sequence at least 85%, preferably at least90%, more preferably at least 95% identical thereto, and the constantdomain of the light chain of this monoclonal antibody or antigen-bindingportion thereof comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95% identical thereto. More preferably, the constantdomain of the heavy chain comprises the amino acid sequence of SEQ IDNo: 29, or an amino acid sequence at least 98%, preferably at least 99%identical thereto, and the constant domain of the light chain comprisesthe amino acid sequence of SEQ ID No: 32, or an amino acid sequence atleast 98%, preferably at least 99% identical thereto. Still morepreferably, the constant domain of the heavy chain comprises the aminoacid sequence of SEQ ID No: 29, and the constant domain of the lightchain comprises the amino acid sequence of SEQ ID No: 32.

Further, a monoclonal antibody capable of binding to human GDF-15, or anantigen-binding portion thereof is provided, wherein the heavy chainvariable domain comprises a CDR3 region comprising the amino acidsequence of SEQ ID NO: 5 or an amino acid sequence that differs by notmore than one amino acid from the amino acid sequence of SEQ ID NO: 5,and wherein the light chain variable domain comprises a CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 7 or an amino acidsequence or an amino acid sequence that differs by not more than oneamino acid from the amino acid sequence of SEQ ID NO: 7. Preferably, theconstant domain of the heavy chain of this monoclonal antibody orantigen-binding portion thereof comprises the amino acid sequence of SEQID No: 29, or an amino acid sequence at least 85%, preferably at least90%, more preferably at least 95% identical thereto, and the constantdomain of the light chain of this monoclonal antibody or antigen-bindingportion thereof comprises the amino acid sequence of SEQ ID No: 32, oran amino acid sequence at least 85%, preferably at least 90%, morepreferably at least 95% identical thereto. More preferably, the constantdomain of the heavy chain comprises the amino acid sequence of SEQ IDNo: 29, or an amino acid sequence at least 98%, preferably at least 99%identical thereto, and the constant domain of the light chain comprisesthe amino acid sequence of SEQ ID No: 32, or an amino acid sequence atleast 98%, preferably at least 99% identical thereto. Still morepreferably, the constant domain of the heavy chain comprises the aminoacid sequence of SEQ ID No: 29, and the constant domain of the lightchain comprises the amino acid sequence of SEQ ID No: 32.

In a second embodiment in accordance with the above embodiments, theheavy chain variable domain of the monoclonal antibody orantigen-binding portion thereof comprises a CDR3 region comprising theamino acid sequence of SEQ ID NO: 5, or the light chain variable domaincomprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7.

In a third embodiment in accordance with the above embodiments, theheavy chain variable domain of the monoclonal antibody orantigen-binding portion thereof comprises a CDR3 region comprising theamino acid sequence of SEQ ID NO: 5, and the light chain variable domaincomprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7.

In a fourth embodiment in accordance with the above embodiments, theheavy chain variable domain of the monoclonal antibody orantigen-binding portion thereof comprises a CDR1 region comprising theamino acid sequence of SEQ ID NO: 3 and a CDR2 region comprising theamino acid sequence of SEQ ID NO: 4, and the light chain variable domainof the monoclonal antibody or antigen-binding portion thereof comprisesa CDR1 region comprising the amino acid sequence of SEQ ID NO: 6 and aCDR2 region comprising the amino acid sequence ser-ala-ser.

In a fifth embodiment in accordance with the above embodiments, theantibody is a humanized antibody. Preferably, all of the variabledomains of the humanized antibody are humanized variable domains.

In a further embodiment in accordance with the above embodiments, theheavy chain variable domain of the monoclonal antibody orantigen-binding portion thereof comprises the amino acid sequence of SEQID No: 28, or an amino acid sequence at least 90%, preferably at least95%, more preferably at least 98%, still more preferably at least 99%identical thereto, and the light chain variable domain of the monoclonalantibody or antigen-binding portion thereof comprises the amino acidsequence of SEQ ID No: 31, or an amino acid sequence at least 90%,preferably at least 95%, more preferably at least 98%, still morepreferably at least 99% identical thereto. In the most preferred aspectof this embodiment, the heavy chain variable domain comprises the aminoacid sequence of SEQ ID No: 28, and the light chain variable domaincomprises the amino acid sequence of SEQ ID No: 31.

In a further preferred embodiment in accordance with the aboveembodiments, the heavy chain of the monoclonal antibody orantigen-binding portion thereof comprises the amino acid sequence of SEQID No: 27, and the light chain of the monoclonal antibody orantigen-binding portion thereof comprises the amino acid sequence of SEQID No: 30.

In a another preferred embodiment in accordance with the aboveembodiments, the heavy chain variable domain of the monoclonal antibodyor antigen-binding portion thereof comprises the amino acid sequence ofSEQ ID No: 34, or an amino acid sequence at least 75%, more preferablyat least 80%, more preferably at least 85%, more preferably at least90%, more preferably at least 95%, more preferably at least 98%, stillmore preferably at least 99% identical thereto, and the light chainvariable domain of the monoclonal antibody or antigen-binding portionthereof comprises the amino acid sequence of SEQ ID No: 37, or an aminoacid sequence at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 95%, more preferablyat least 98%, still more preferably at least 99% identical thereto. Inthe most preferred aspect of this embodiment in accordance with theabove embodiments, the heavy chain variable domain comprises the aminoacid sequence of SEQ ID No: 34, and the light chain variable domaincomprises the amino acid sequence of SEQ ID No: 37.

In still another embodiment in accordance with the above first to thirdembodiment, the heavy chain variable domain comprises a regioncomprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region andcomprising the amino acid sequence of SEQ ID NO: 1 or a sequence 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto,and the light chain variable domain comprises a region comprising anFR1, a CDR1, an FR2, a CDR2 and an FR3 region and comprising the aminoacid sequence of SEQ ID NO: 2 or a sequence 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical thereto.

In a preferred embodiment in accordance with the above first to thirdembodiment, the heavy chain variable domain comprises a regioncomprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region andcomprising the amino acid sequence of SEQ ID NO: 1 or a sequence 95%identical thereto, and the light chain variable domain comprises aregion comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region andcomprising the amino acid sequence of SEQ ID NO: 2 or a sequence 95%identical thereto.

In a more preferred embodiment in accordance with the above first tothird embodiment, the heavy chain variable domain comprises a regioncomprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region andcomprising the amino acid sequence of SEQ ID NO: 1 or a sequence 98%identical thereto, and the light chain variable domain comprises aregion comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region andcomprising the amino acid sequence of SEQ ID NO: 2 or a sequence 98%identical thereto.

In a still more preferred embodiment in accordance with the above firstto third embodiment, the heavy chain variable domain comprises a regioncomprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region andcomprising the amino acid sequence of SEQ ID NO: 1, and the light chainvariable domain comprises a region comprising an FR1, a CDR1, an FR2, aCDR2 and an FR3 region and comprising the amino acid sequence of SEQ IDNO: 2.

Further, a monoclonal antibody capable of binding to human GDF-15, or anantigen-binding portion thereof is provided, wherein the heavy chainvariable domain comprises a CDR1 region comprising the amino acidsequence of SEQ ID NO: 3 and a CDR2 region comprising the amino acidsequence of SEQ ID NO: 4, and wherein the light chain variable domaincomprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:6 and a CDR2 region comprising the amino acid sequence of SEQ ID NO: 7.In a preferred aspect of this embodiment, the antibody may have CDR3sequences as defined in any of the embodiments of the inventiondescribed above.

In another embodiment, the a monoclonal antibody capable of binding tohuman GDF-15, or an antigen-binding portion thereof is provided, whereinthe antibody or antigen-binding portion thereof is capable of inhibitingcancer growth in a mammal, preferably a human patient.

In another embodiment in accordance with the above embodiments, theinvention relates to an antigen-binding portion capable of binding tohuman GDF-15, wherein the antigen-binding portion is a single-domainantibody (also referred to as “Nanobody™”). In one aspect of thisembodiment, the single-domain antibody comprises the CDR1, CDR2, andCDR3 amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:5, respectively. In another aspect of this embodiment, the single-domainantibody comprises the CDR1, CDR2, and CDR3 amino acid sequences of SEQID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 7, respectively. In a preferredaspect of this embodiment, the single-domain antibody is a humanizedantibody.

Preferably, the antibodies of the invention capable of binding to humanGDF-15 or the antigen-binding portions thereof have an equilibriumdissociation constant for human GDF-15 that is equal to or less than 100nM, less than 20 nM, preferably less than 10 nM, more preferably lessthan 5 nM and most preferably between 0.1 nM and 2 nM.

In another embodiment of the invention, the antibody capable of bindingto human GDF-15 or the antigen-binding portion thereof binds to the samehuman GDF-15 epitope as the antibody to human GDF-15 obtainable from thecell line B1-23 deposited with the Deutsche Sammlung für Mikroorganismenund Zellkulturen GmbH (DMSZ) under the accession No. DSM ACC3142. Asdescribed herein, antibody binding to human GDF-15 in accordance withthe present invention is assessed by surface plasmon resonancemeasurements as a reference standard method, in accordance with theprocedures described in Example 1. Binding to the same epitope on humanGDF-15 can be assessed similarly by surface plasmon resonancecompetitive binding experiments of the antibody to human GDF-15obtainable from the cell line B1-23 and the antibody that is expected tobind to the same human GDF-15 epitope as the antibody to human GDF-15obtainable from the cell line B1-23.

In a very preferred embodiment, the antibody is the monoclonal antibodycapable of binding to human GDF-15 obtainable from the cell line B1-23deposited with the Deutsche Sammlung für Mikroorganismen undZellkulturen GmbH (DMSZ) under the accession No. DSM ACC3142 or anantigen-binding portion thereof.

In a preferred embodiment, the antibody capable of binding to humanGDF-15 or the antigen-binding portion thereof according to the inventionis a humanized monoclonal antibody or an antigen-binding portionthereof. For any given non-human antibody sequence in accordance withthe invention (i.e. a donor antibody sequence), humanized monoclonalanti-human-GDF-15 antibodies of the invention or antigen-bindingportions thereof can be generated in accordance with techniques known inthe art, as described above.

In a very preferred embodiment, the monoclonal antibody capable ofbinding to human GDF-15 or antigen-binding portion thereof is ahumanized antibody derived from the monoclonal antibody capable ofbinding to human GDF-15 obtainable from the cell line B1-23 depositedwith the Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH(DMSZ) under the accession No. DSM ACC3142, or an antigen-bindingportion thereof. In a non-limiting aspect of this embodiment, the heavychain variable domain of the humanized antibody or antigen-bindingportion thereof comprises a CDR3 region comprising the amino acidsequence of SEQ ID NO: 5, and the light chain variable domain of thehumanized antibody or antigen-binding portion thereof comprises a CDR3region comprising the amino acid sequence of SEQ ID NO: 7. In a furthernon-limiting aspect of this embodiment, the heavy chain variable domainof the humanized antibody or antigen-binding portion thereof comprisesor further comprises a CDR1 region comprising the amino acid sequence ofSEQ ID NO: 3 and a CDR2 region comprising the amino acid sequence of SEQID NO: 4, and the light chain variable domain of the humanized antibodyor antigen-binding portion thereof comprises or further comprises a CDR1region comprising the amino acid sequence of SEQ ID NO: 6 and a CDR2region comprising the amino acid sequence of SEQ ID NO: 7.

Further, a monoclonal antibody capable of binding to human GDF-15, or anantigen-binding portion thereof is provided, wherein the binding isbinding to a conformational or discontinuous epitope on human GDF-15comprised by the amino acid sequences of SEQ ID No: 25 and SEQ ID No:26. In a preferred aspect of this embodiment, the antibody orantigen-binding portion thereof is an antibody or antigen-bindingportion thereof as defined in any one of the above embodiments.

In another embodiment of the invention in accordance with the aboveembodiments, the antibody capable of binding to human GDF-15 or theantigen-binding portion thereof is a diabody. In one aspect of thisembodiment, the diabody is bivalent and monospecific, with two identicalantigen binding sites for human GDF-15. In a second, alternative aspectof this embodiment, the diabody is bivalent and bispecific, with oneantigen binding site being a binding site for human GDF-15, and theother antigen binding site being a binding site for a different antigen.Non-limiting examples for the different antigen according to this secondaspect of this embodiment are i) cell surface antigens that areco-expressed with GDF-15 at high levels on the same cancer (e.g. athigher levels compared to a control sample of the same patient obtainedfrom a non-cancerous part of the tissue which is the tissue of origin ofthe cancer), and ii) cell surface antigens on cells of the immune systemwhich are known as useful antigens for the recruitment of cells of theimmune system to the tumor.

In still another embodiment of the invention in accordance with theabove embodiments, the antibody capable of binding to human GDF-15 orthe antigen-binding portion thereof is linked to a drug. In non-limitingaspects of this embodiment, the drug can be a known anticancer agentand/or an immune-stimulatory molecule. Known anticancer agents includealkylating agents such as cisplatin, carboplatin, oxaliplatin,mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide;anti-metabolites such as azathioprine and mercaptopurine; alkaloids suchas vinca alkaloids (e.g. vincristine, vinblastine, vinorelbine, andvindesine), taxanes (e.g. paclitaxel, docetaxel) etoposide andteniposide; topoisomerase inhibitors such as camptothecins (e.g.irinotecan and topotecan); cytotoxic antibiotics such as actinomycin,anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin,epirubicin, bleomycin, plicamycin and mitomycin; and radioisotopes.Linking of the antibodies or the antigen-binding portions thereof of theinvention to anticancer agents is expected to result in stronger cancertumor growth inhibition compared to the antibody without the anticanceragent, because the resulting conjugate will accumulate at the site ofthe tumor due to the presence of GDF-15 in the tumor, leading to theaccumulation of the anticancer agent at the site of the tumor and toenhanced effects of the anticancer agent on the tumor.

In a further embodiment in accordance with the above embodiments, theantibody capable of binding to human GDF-15 or the antigen-bindingportion thereof is modified by an amino acid tag. Non-limiting examplesof such tags include Polyhistidin (His-) tags, FLAG-tag, Hemagglutinin(HA) tag, glycoprotein D (gD) tag, and c-myc tag. Tags may be used forvarious purposes. For instance, they may be used to assist purificationof the antibody capable of binding to human GDF-15 or theantigen-binding portion thereof, or they may be used for detection ofthe antibody or the antigen-binding portion thereof (e.g. when used indiagnostic assays). Preferably, such tags are present at the C-terminusor N-terminus of the antibody capable of binding to human GDF-15 or theantigen-binding portion thereof.

In a preferred embodiment of the present invention in accordance withthe above embodiments, the antibody capable of binding to human GDF-15or the antigen-binding portion thereof is capable of inhibiting cancergrowth in a mammal, preferably a human patient.

In another preferred embodiment of the present invention in accordancewith the above embodiments, the human GDF-15 is recombinant human.GDF-15 having the amino acid sequence represented by SEQ ID No: 8.

In still another preferred embodiment of the present invention inaccordance with the above embodiments, the binding of the antibodycapable of binding to human GDF-15 or the antigen-binding portionthereof is a binding to conformational or discontinuous epitope on humanGDF-15.

Preferably, the monoclonal antibodies of the present invention capableof binding to human GDF-15 or the antigen-binding portions thereof areisolated antibodies.

In a preferred embodiment of the above antibodies or antigen-bindingportions thereof according to the invention, the antibody has a size ofmore than 100 kDa, preferably more than 110 kDa, more preferably morethan 120 kDa, still more preferably more than 130 kDa, and mostpreferably more than 140 kDa. Preferably, the antibody is a full-lengthantibody, more preferably a full-length IgG antibody.

The invention also relates to an expression vector comprising anucleotide sequence encoding the antibody or antigen-binding portionthereof as defined above.

Further, the present invention also provides a cell line capable ofproducing an antibody or antigen-binding portion thereof according tothe present invention.

In one embodiment, the cell line can be derived from any cell line thatis known in that art and suitable for the production of antibodies orantigen-binding portions thereof.

In a preferred embodiment, the cell line is the cell line B1-23deposited with the Deutsche Sammlung für Mikroorganismen undZellkulturen GmbH (DMSZ) under the accession No. DSM ACC3142.

In another preferred embodiment, the cell line contains an expressionvector according to the invention as defined above.

B) PHARMACEUTICAL COMPOSITIONS

In a further embodiment, the present invention relates to apharmaceutical composition comprising any of the antibodies orantigen-binding portions thereof as defined above.

Pharmaceutical compositions in accordance with the present invention areprepared in accordance with known standards for the preparation ofpharmaceutical compositions containing antibodies and portions thereof.

For instance, the compositions are prepared in a way that they can bestored and administered appropriately, e.g. by using pharmaceuticallyacceptable components such as carriers, excipients or stabilizers.

Such pharmaceutically acceptable components are not toxic in the amountsused when administering the pharmaceutical composition to a patient. Thepharmaceutical acceptable components added to the pharmaceuticalcompositions may depend on the particular intended use of thepharmaceutical compositions and the route of administration.

In general, the pharmaceutically acceptable components used inconnection with the present invention are used in accordance withknowledge available in the art, e.g. from Remington's PharmaceuticalSciences, Ed. A R Gennaro, 20th edition, 2000, Williams & Wilkins, PA,USA.

C) THERAPEUTIC METHODS AND PRODUCTS FOR USE IN THESE METHODS

The present invention further relates to a method for treating cancercachexia in a mammal. The method comprises administering an antibody orantigen-binding portion thereof as defined above, or a pharmaceuticalcomposition as defined above to said mammal. Alternatively, the presentinvention relates to an antibody or antigen-binding portion thereof asdefined above, or a pharmaceutical composition as defined above for usein these methods. In a very preferred aspect of these embodiments, themammal is a human patient.

The present invention further relates to a method for treating cancer ina mammal. The method comprises administering an antibody orantigen-binding portion thereof as defined above, or a pharmaceuticalcomposition as defined above to said mammal. Alternatively, the presentinvention relates to an antibody or antigen-binding portion thereof asdefined above, or a pharmaceutical composition as defined above for usein these methods. In a very preferred aspect of these embodiments, themammal is a human patient.

When taking into account the present invention, it becomes clear thatthe anti-GDF-15 antibodies known from WO 2005/099746, WO 2009/021293 andJohnen H et al., Nature Medicine, 2007 only inhibit cancer-inducedweight loss, but fail to inhibit other effects of human GDF-15 such asthose related to cancer growth.

The present invention relates to several surprising advantages comparedto the effects observed in the art.

In particular, one main benefit of the invention lies in that theanti-GDF-15 antibodies disclosed herein can be used to more effectivelytreat cancer-induced weight loss and/or cancer cachexia.

For instance, the treatment with the antibodies according to theinvention can completely prevent cancer cachexia (when givenprophylactically) or completely reverse cancer cachexia (when givenafter the onset of cancer cachexia).

Moreover, the antibodies according to the invention can even increasethe body weight of the treated mammal during a prophylactic treatmentfor the prevention of cachexia. Likewise, it is expected that in thecourse of a therapeutic treatment started after the onset of cancercachexia, the antibodies according to the invention can not only reversethe loss in body weight, but also increase the body weight of thetreated mammal compared to its body weight before the onset of cancercachexia.

This unexpected effect of the antibodies according to the invention maybe beneficial in various clinical situations. For instance,administration of many ingredients that are pharmaceutically activeagainst cancer (e.g. various chemotherapeutic drugs) can lead to a lossof body weight of mammals including human patients. Such an additionalloss in body weight could be counteracted by the increase in body weightdue to the administration of the antibodies according to the invention.Therefore, the uses of the antibodies according to the invention may beparticularly advantageous and safe for combination regimens withadditional chemotherapeutic drugs. Similarly, the uses of the antibodiesaccording to the invention may be particularly advantageous for mammalssuch as human patients that already had a low body weight prior to theonset of cancer and/or prior to the onset of cancer cachexia. Patientswith a low body weight may for instance be cachectic patients, e.g.patients with a body-mass-index of less than 18 kg/m².

Moreover, unexpectedly, according to the invention, the antibodies arenot only effective for the treatment of cancer cachexia, but alsoeffective for the treatment of cancer.

Thus, the treatment methods and products for use of the antibodiesaccording to the invention are expected to be particularly beneficialfor the treatment of cancer patient sub-groups which suffer fromcancer-induced weight loss and/or cancer cachexia, respectively.

However, the effects according to the invention are also expected to beadvantageous for the treatment of a complete patient group of a cancerreferred to herein: By using the antibodies according to the inventionthat are effective both against the cancer itself and againstcancer-induced weight loss and/or cancer cachexia, cancer and cancercachexia treatments may be simplified by using the same treatment forall cancer patients, irrespective of whether or not they suffer fromcancer-induced weight loss and/or cancer cachexia. This is because dueto the dual effects of the antibodies against cancer and cancercachexia, it is expected that these antibodies will obviate the need foradditional drugs for the treatment of cancer cachexia.

Likewise, due to the dual effects of the antibodies in accordance withthe invention, it may also become unnecessary to diagnose cancer-inducedweight loss and/or cancer cachexia. Hence it is expected that theoverall costs of therapy and diagnosis will be reduced.

Therefore, in a preferred embodiment of the above methods, orantibodies, antigen-binding portions thereof or pharmaceuticalcompositions for use in these methods according to the invention, themethod for treating cancer cachexia is a method for completelypreventing or completely reverting cancer cachexia. In a more preferredembodiment of this method, or the antibodies, antigen-binding portionsthereof or pharmaceutical compositions for use in this method, themethod for treating cancer cachexia is a method for completelypreventing cancer cachexia. In an alternative more preferred embodimentof this method, or the antibodies, antigen-binding portions thereof orpharmaceutical compositions for use in this method, the method fortreating cancer cachexia is a method for completely reverting cancercachexia.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods according to the invention, only mammals suffering fromboth

-   i) the cancer, and-   ii) cancer cachexia    -   are treated in the method.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods according to the invention, the method increases bodyweight of the mammal compared to its body weight before the onset ofcancer cachexia. Preferably, the increase in body weight of the mammalis at least 1.5%, preferably at least 2.5%, more preferably at least 5%compared to its body weight before the onset of cancer cachexia.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods according to the invention, the method is a method forboth treating cancer and treating cancer cachexia in the same mammal.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods according to the invention, the antibody has a size ofmore than 100 kDa, preferably more than 110 kDa, more preferably morethan 120 kDa, still more preferably more than 130 kDa, and mostpreferably more than 140 kDa. Preferably, the antibody is a full-lengthantibody, more preferably a full-length IgG antibody.

In a further preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods according to the invention, the antibody has an Fcportion which is capable of binding to the Fc receptor.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods according to the invention, the cancer cells of themammal endogenously express GDF-15 and/or the cancer cells of the mammalstimulate endogenous expression of GDF-15 in non-cancerous cells of themammal.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods according to the invention, the cancer cells of themammal are characterized in that they endogenously express GDF-15.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods according to the invention, the mammal is humanpatient.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods according to the invention, the human GDF-15 isrecombinant human GDF-15 having the amino acid sequence represented bySEQ ID No: 8.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods, the human patient has elevated GDF-15 levels in bloodserum before administration. In a patient sub-group having elevatedGDF-15 levels in blood serum, the treatment methods according to theinvention are expected to be particularly effective at inhibiting cancergrowth. In the most preferred aspect of this embodiment, GDF-15 levelsare GDF-15 protein levels measured using the antibody according to theinvention obtainable from the hybridoma cell line B1-23 deposited withthe Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (DSMZ)under the accession No. DSM ACC3142, preferably measured byimmunochemistry.

In another embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods, the antibody or antigen-binding portion thereof is thesole ingredient pharmaceutically active against cancer used in themethod.

In an alternative embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods, the antibody or antigen-binding portion thereof isused in combination with one or more further ingredientspharmaceutically active against cancer. In one aspect of thisembodiment, the one or more further ingredients pharmaceutically activeagainst cancer is a known anticancer agent and/or an immune-stimulatorymolecule as defined above. Thus, the anticancer agent can for instancebe selected from alkylating agents such as cisplatin, carboplatin,oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, andifosfamide; anti-metabolites such as azathioprine and mercaptopurine;alkaloids such as vinca alkaloids (e.g. vincristine, vinblastine,vinorelbine, and vindesine), taxanes (e.g. paclitaxel, docetaxel)etoposide and teniposide; topoisomerase inhibitors such as camptothecins(e.g. irinotecan and topotecan); cytotoxic antibiotics such asactinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicin,idarubicin, epirubicin, bleomycin, plicamycin and mitomycin; andradioisotopes. Due to the increasing effect of the antibodies accordingto the invention on body weight of the mammals including human patients,these combined uses of the antibodies or antigen-binding portionsthereof and the ingredients pharmaceutically active against cancer areexpected to be particularly safe, because they may compensate a possibleadditional weight loss resulting from the administration of theingredients pharmaceutically active against cancer.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods, the cancer is selected from the group consisting ofbrain cancers including glioma, cancers of the nervous system, melanoma,lung cancer, lip and oral cavity cancer, hepatic carcinoma, leukemia,Hodgkin lymphoma, Non-Hodgkin lymphoma, bladder cancer, cervix utericancer, corpus uteri cancer, testis cancer, thyroid cancer, kidneycancer, gallbladder cancer, multiple myeloma, nasopharynx cancer, larynxcancer, pharynx cancer, oesophagus cancer, gastrointestinal tumorsincluding stomach and colorectal cancer, pancreatic cancer, prostatecancer, ovarian cancer and breast cancer, preferably from the groupconsisting of melanoma, prostate cancer, breast cancer, brain cancersincluding glioma, colorectal cancer, stomach cancer, oesophagus cancerand ovarian cancer, and most preferably is melanoma. In one embodimentthe cancer is selected from the above group, which further comprisesendometrial cancer, such as endometrial carcinoma, breast cancerincluding subtypes of breast cancer, in particular triple-negativebreast cancer and bladder cancer such as urothelial cell carcinoma.

In another preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods, the tumor or tumors formed by the cancer have higherhuman GDF-15 levels prior to administration compared to a control sampleof the same patient obtained from a non-cancerous part of the tissuewhich is the tissue of origin of the cancer, preferably 1.2-fold higherlevels, more preferably 1.5-fold higher levels, still more preferably2-fold higher levels and most preferably 5-fold higher levels. In apatient sub-group having higher GDF-15 levels in the tumor or tumorsformed by the cancer compared to the above control sample, the treatmentmethods according to the invention are expected to be particularlyeffective at inhibiting cancer growth.

In a very preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods, the method for treating cancer comprises inhibitingcancer growth. In a preferred aspect of this embodiment, cancer growthis stopped. In a more preferred aspect, the cancer shrinks.

In a preferred embodiment of the above methods, or antibodies,antigen-binding portions thereof or pharmaceutical compositions for usein these methods, the method for treating cancer comprises the inductionof killing of cancer cells by NK cells and CD8+ T cells in the humanpatient. Due to their capability of preventing GDF-15 mediateddown-regulation of the known immune surveillance regulator NKG2D, theantibodies or antigen-binding portions thereof according to theinvention are expected to restore immune surveillance and induce thekilling of cancer cells by NK cells and CD8+ T cells, in addition toeffects of the antibodies or antigen-binding portions thereof that areindependent of the immune system.

D) KITS

The present invention also provides kits comprising the pharmaceuticalcompositions as defined above.

In one embodiment, the kits are kits for use in the methods according tothe invention as defined above.

In further embodiments, the present invention also provides a diagnostickit comprising any of the antibodies or antigen-binding portions thereofaccording to the invention.

In one embodiment, the diagnostic kit may be used to detect whether thetumor or tumors of a cancer patient formed by the cancer have higherhuman GDF-15 levels compared to a control sample of the same patientobtained from a non-cancerous part of the tissue which is the tissue oforigin of the cancer.

In another embodiment, the diagnostic kit may be used to detect whethera human cancer patient has elevated GDF-15 levels in blood serum.

E) SEQUENCES

The amino acid sequences referred to in the present application are asfollows (in an N-terminal to C-terminal order; represented in theone-letter amino acid code)

SEQ ID No: 1 (Region of the Heavy Chain VariableDomain comprising an FR1, a CDR1, an FR2, a CDR2and an FR3 region from the Polypeptide Sequenceof monoclonal anti-human GDF-15 mAb-B1-23):QVKLQQSGPGILQSSQTLSLTCSFSGFSLSTSGMCVSWIRQPSGKGLEWLAHIYWDDDKRYNPTLKSRLTISKDPSRNQVFLKITSVDTADTATYYCSEQ ID No: 2 (Region of the Light Chain VariableDomain comprising an FR1, a CDR1, an FR2, a CDR2and an FR3 region from the Polypeptide Sequenceof monoclonal anti-human GDF-15 mAb-B1-23):DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWFLQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCSEQ ID No: 3 (Heavy Chain CDR1 Region PeptideSequence of monoclonal anti-human GDF-15 mAb-B1-23): GFSLSTSGMGSEQ ID No: 4 (Heavy Chain CDR2 Region PeptideSequence of monoclonal anti-human GDF-15 mAb-B1-23): IYWDDDKSEQ ID No: 5 (Heavy Chain CDR3 Region PeptideSequence of monoclonal anti-human GDF-15 mAb-B1-23): ARSSYGAMDYSEQ ID No: 6 (Light Chain CDR1 Region PeptideSequence of monoclonal anti-human GDF-15 mAb-B1-23): QNVGTNLight Chain CDR2 Region Peptide Sequence of monoclonal anti-human GDF-15 mAb-B1-23: SASSEQ ID No: 7 (Light Chain CDR3 Region PeptideSequence of monoclonal anti-human GDF-15 mAb-B1-23): QQYNNFPYTSEQ ID No: 8 (recombinant mature human GDF-15 protein):GSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGV SLQTYDDLLAKDCHCISEQ ID No: 9 (human GDF-15 precursor protein):MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSL QTYDDLLAKDCHCISEQ ID No: 10 (human GDF-15 precursor protein +N-terminal and C-terminal GSGS linker):GSGSGSGMPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTALVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIGSGSGSG SEQ ID No: 11 (Flag peptide): DYKDDDDKGGSEQ ID No: 12 (HA peptide): YPYDVPDYAGSEQ ID No: 13 (peptide derived from human GDF-15): ELHLRPQAARGRRSEQ ID No: 14 (peptide derived from human GDF-15): LHLRPQAARGRRRSEQ ID No: 15 (peptide derived from human GDF-15): HLRPQAARGRRRASEQ ID No: 16 (peptide derived from human GDF-15): LRPQAARGRRRARSEQ ID No: 17 (peptide derived from human GDF-15): RPQAARGRRRARASEQ ID No: 18 (peptide derived from human GDF-15): PQAARGRRRARARSEQ ID No: 19 (peptide derived from human GDF-15): QAARGRRRARARNSEQ ID No: 20 (peptide derived from human GDF-15): MHAQIKTSLHRLKSEQ ID No: 25 (GDF-15 peptide comprising part ofthe GDF-15 Epitope that binds to B1-23): EVQVTMCIGACPSQFRSEQ ID No: 26 (GDF-15 peptide comprising part ofthe GDF-15 Epitope that binds to B1-23): TDTGVSLQTYDDLLAKDCHCI

The nucleic acid sequences referred to in the present application are asfollows (in a 5′ to 3′ order; represented in accordance with thestandard nucleic acid code):

SEQ ID No: 21 (DNA nucleotide sequence encoding the amino acid sequence defined in SEQ ID No: 1):CAAGTGAAGCTGCAGCAGTCAGGCCCTGGGATATTGCAGTCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGTACTTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTATAACCCAACCCTGAAGAGCCGGCTCACAATCTCCAAGGATCCCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACATACTACTGTSEQ ID No: 22 (DNA nucleotide sequence encodingthe amino acid sequence defined in SEQ ID No: 2):GACATTGTGCTCACCCAGTCTCCAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTGGCCTGGTTTCTACAGAAACCAGGGCAATCTCCTAAAGCACTTATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAACGTGCAGTCTGAAGA CTTGGCAGAGTATTTCTGTSEQ ID No: 23 (DNA nucleotide sequence encodingthe amino acid sequence defined in SEQ ID No: 5):GCTCGAAGTTCCTACGGGGCAATGGACTACSEQ ID No: 24 (DNA nucleotide sequence encodingthe amino acid sequence defined in SEQ ID No: 7):CAGCAATATAACAACTTTCCGTACACGSEQ ID No: 27 (amino acid sequence of the heavychain of the H1L5 humanized B1-23 anti-GDF-15  antibody):QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYNPTLKSRLTITKDPSKNQVVLTMTNMDPVDTATYYCARSSYGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKSEQ ID No: 28 (amino acid sequence of the heavychain variable domain of the H1L5 humanized B1-23 anti-GDF-15 antibody):QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYNPTLKSRLTITKDPSKNQVVLTMTNMDPVDTATYYCA RSSYGAMDYWGQGTLVTVSSSEQ ID No: 29 (amino acid sequence of the heavychain constant domain of the H1L5 humanized B1-23 anti-GDF-15 antibody):ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID No: 30 (amino acid sequence of the lightchain of the H1L5 humanized B1-23 anti-GDF-15 antibody):DIVLTQSPSFLSASVGDRVTITCKASQNVGTNVAWFQQKPGKSPKALIYSASYRYSGVPDRFTGSGSGTEFTLTISSLQPEDFAAYFCQQYNNFPYTFGGGTKLEIKRAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECSEQ ID No: 31 (amino acid sequence of the lightchain variable domain of the H1L5 humanized B1-23 anti-GDF-15 antibody):DIVLTQSPSFLSASVGDRVTITCKASQNVGTNVAWFQQKPGKSPKALIYSASYRYSGVPDRFTGSGSGTEFTLTISSLQPEDFAAYFCQQYNNFPYTF GGGTKLEIKRSEQ ID No: 32 (amino acid sequence of the light chain constant domain of the H1L5 humanized B1-23 anti-GDF-15 antibody): APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGECSEQ ID No: 33 (amino acid sequence of the heavychain of the chimeric B1-23 anti-GDF-15 antibody):QVKLQQSGPGILQSSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPTLKSRLTISKDPSRNQVFLKITSVDTADTATYYCARSSYGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKSEQ ID No: 34 (amino acid sequence of the heavychain variable domain of the chimeric B1-23 anti- GDF-15 antibody):QVKLQQSGPGILQSSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPTLKSRLTISKDPSRNQVFLKITSVDTADTATYYCA RSSYGAMDYWGQGTSVTVSSSEQ ID No: 35 (amino acid sequence of the heavy chain constant domain of the chimeric B1-23 anti- GDF-15 antibody):ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID No: 36 (amino acid sequence of the light chain of the chimeric B1-23 anti-GDF-15  antibody):DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWFLQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID No: 37 (amino acid sequence of the lightchain variable domain of the chimeric B1-23 anti- GDF-15 antibody):DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWFLQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNFPYTF GGGTKLEIKRTVASEQ ID No: 38 (amino acid sequence of the light chain constant domain of the chimeric B1-23 anti- GDF-15 antibody):APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC

F) EXAMPLES

The present invention is illustrated by the following non-limitingExamples:

Example 1: Generation and Characterization of the Murine GDF-15 AntibodyB1-23, and Generation of Chimeric and Humanized Antibodies

The antibody B1-23 was generated in a GDF-15 knock out mouse.Recombinant human GDF-15 (SEQ ID No: 8) was used as the immunogen.

The hybridoma cell line B1-23 producing mAb-B1-23 was deposited with theDeutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (DMSZ) underthe accession No. DSM ACC3142, in accordance with the Budapest Treaty.

By means of a commercially available test strip system, B1-23 wasidentified as an IgG2a (kappa chain) isotype. Using surface plasmonresonance measurements, the dissociation constant (Kd) was determined asfollows:

Binding of the monoclonal anti-human-GDF-15 antibody anti-human GDF-15mAb-B1-23 was measured by employing surface plasmon resonancemeasurements using a Bio-Rad® ProteOn™ XPR36 system and Bio-Rad® GLCsensor chips:

For preparing the biosensors recombinant mature human GDF-15 protein wasimmobilized on flow cells 1 and 2. On one flow cell recombinant GDF-15derived from Baculvirus-transfected insect cells (HighFive insect cells)and on the other recombinant protein derived from expression in E. coliwas used. The GLC sensor chip was activated using Sulfo-NHS(N-Hydroxysulfosuccinimide) and EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (Bio-Rad®ProteOn™ Amine Coupling Kit) according to the manufacturer'srecommendation, the sensor surface was subsequently loaded with theproteins up to a density of about 600 RU (1 Ru=1 pg mm⁻²). Thenon-reacted coupling groups were then quenched by perfusion with 1Methanolamine pH 8.5 and the biosensor was equilibrated by perfusing thechip with running buffer (10M HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005%Tween®20, pH 7.4, referred to as HBS150). As controls two flow cellswere used, one empty with no protein coupled and one coupled with annon-physiological protein partner (human Interleukin-5), which wasimmobilized using the same coupling chemistry and the same couplingdensity. For interaction measurements anti-human GDF-15 mAb-B1-23 wasdissolved in HBS150 and used in six different concentrations as analyte(concentration: 0.4, 0.8, 3, 12, 49 and 98 nM). The analyte was perfusedover the biosensor using the one-shot kinetics setup to avoidintermittent regeneration, all measurements were performed at 25° C. andusing a flow rate of 100 μl min⁻¹. For processing the bulk face effectand unspecific binding to the sensor matrix was removed by subtractingthe SPR data of the empty flow cell (flow cell 3) from all other SPRdata. The resulting sensogram was analyzed using the software ProteOnManager version 3.0. For analysis of the binding kinetics a 1:1Langmuir-type interaction was assumed. For the association rate constanta value of 5.4±0.06×10⁵ M⁻¹s⁻¹ (k_(on)) and for the dissociation rateconstant a value of 4.3±0.03×10⁻⁴ s⁻¹ (k_(off)) could be determined(values are for the interaction of anti-human GDF-15 mAb-B1-23 withGDF-15 derived from insect cell expression). The equilibriumdissociation constant was calculated using the equationK_(D)=k_(off)/k_(on) to yield a value of about 790 pM. Affinity valuesfor the interaction of GDF-15 derived from E. coli expression and theanti-human GDF-15 mAb-B1-23 differ by less than a factor of 2, rateconstants for GDF-15 derived from insect cells and E. coli deviate byabout 45% and are thus within the accuracy of SPR measurements andlikely do not reflect a real difference in affinity. Under theconditions used the anti-human GDF-15 mAb-B1-23 shows no binding tohuman interleukin-5 and thus confirms the specificity of the interactiondata and the anti-human GDF-15 mAb-B1-23.

The amino acid sequence of recombinant human GDF-15 (as expressed inBaculovirus-transfected insect cells) is:

(SEQ ID No: 8) GSARNGDHCP LGPGRCCRLH TVRASLEDLG WADWVLSPREVQVTMCIGAC PSQFRAANMH AQIKTSLHRL KPDTVPAPCCVPASYNPMVL IQKTDTGVSL QTYDDLLAKD CHCI

Thus, using surface plasmon resonance measurements, the dissociationconstant (Kd) of 790 pM was determined. As a comparison: thetherapeutically used antibody Rituximab has a significantly loweraffinity (Kd=8 nM).

From the murine anti-human GDF-15 mAb-B1-23, a chimeric anti-humanGDF-15 mAb-B1-23 antibody according to the invention was generated byreplacing constant domains of the murine antibody with the constantdomains of a human IgG1 antibody (trastuzumab backbone). The amino acidsequence of the heavy chain of this chimeric antibody is shown in SEQ IDNo: 33, and the amino acid sequence of the light chain of this chimericantibody is shown in SEQ ID No: 36.

From the chimeric anti-human GDF-15 mAb-B1-23, a humanized anti-humanGDF-15 mAb-B1-23 antibody according to the invention was developed byhumanizing the variable domains of the chimeric antibody, i.e. byreplacing the framework regions of the chimeric antibody with humansequences. The amino acid sequence of the heavy chain of this humanizedantibody is shown in SEQ ID No: 27, and the amino acid sequence of thelight chain of this humanized antibody is shown in SEQ ID No: 30. Thisantibody is referred to as H1L5 anti-GDF-15 antibody or humanizedB1-23-H1L5 antibody or H1L5 antibody.

In order to generate the above-mentioned chimeric anti-human. GDF-15mAb-B1-23 antibody and the humanized B1-23-H1L5 antibody as indicatedabove, the cDNAs encoding the antibody sequences were optimized, and thegenes were synthesized. The gene sequences were then cloned into acloning/expression vector system. From these vectors, plasmid DNA withlow endotoxin levels was synthesized.

The plasmid DNA was then transiently transfected into CHO cells,followed by an analysis and quantification of antibody expression usinga protein A biosensor. The cDNA of candidate cultures for antibodyexpression was sequenced. The obtained monoclonal antibodies wereanalyzed (see Examples 7 to 9).

Example 2: Antagonization of GDF-15 Mediated Effects with mAB B1-23

a) The NKG2D (Natural Killer Group 2D) receptor, which is expressed onNK cells and CD8+ T cells, is known to play an important role in theimmune surveillance against tumors. Transformed as well as viralinfected cells express ligands, which bind to the NKG2D receptor,thereby activating the cytotoxic effector functions of the describedimmune cells. In that way transformed cells can be detected andeliminated by the immune system. After treatment of immune cells witheither recombinant human GDF-15 or tumor cell secreted GDF-15 in vitrofor 72 hours, the expression level of NKG2D on the cell surface oflymphocytes was downregulated (FIG. 1).

After 72 hours incubation the immune cells were stained with thefollowing FACS-antibodies: anti CD3, anti CD56, anti-NKG2D. Using thisantibody combination, the experiment focused on NK cells and their NKG2Dsurface expression. The low NKG2D level on immune cells led to animpaired tumor/target cell lysis. The GDF-15 mediated downregulation ofNKG2D was prevented by mAb B1-23.

It is therefore concluded that human GDF-15 downregulates expression ofNKG2D on the cell surface of lymphocytes and thereby downregulatesimmune surveillance against tumors. By binding to human GDF-15, theantibodies of the present invention are capable of preventing GDF-15mediated downregulation of NKG2D and should be capable of restoringimmune surveillance and inducing the killing of cancer cells by NK cellsand CD8+ T cells. Given that the CDR regions of the mAb B1-23 antibodycorrespond to CDR regions of the chimeric and humanized antibodies, itis expected that the functional properties including the bindingproperties of these antibodies are similar.

b) The treatment of the ovarian cancer cell line SK-OV-3 withrecombinant GDF-15 led to the phosphorylation of AKT. AKT is a molecule,which is part of the PI3K-pathway and contributes to the activation andproliferation of cells. In this experiment SK-OV-3 cells were treatedwith 10 ng/ml recombinant GDF-15 for 10 min at 37° C., 5% CO2. 5 minutespreincubation of 2 μg mAb-B1-23 with 10 ng/ml GDF-15 at 37° C. blockedthe GDF-15 mediated AKT-phosphorylation (FIG. 2). This showed theneutralizing effect of mAb-B1-23.

c) Treatment of immune cells with recombinant. GDF-15 led to thephosphorylation of JNK, a kinase, which is activated either by cytokinesor by stress. Antagonization of 10 ng/ml GDF-15 with 2 μg mAb-B1-23 (5minute preincubation at 37°) blocked the GDF-15 mediatedJNK1/2-phosphorylation (FIG. 3).

Example 3: Inhibition of Cancer Cell Proliferation Using mAb B1-23

Data generated with B1-23 showed an antiproliferative effect of theantibody on cancer cells in vitro. The strongest antiproliferativeeffect was observed using the prostate cancer cell line LnCap, whichproduces lots of GDF-15. A metabolic assay (Alamar Blue® assay) showed adecrease of proliferation of 30% after 72 hrs when mAb-B1-23 waspresent, compared with the control group, where the antibody was notapplied. Since cytotoxic effects of the antibody have been excluded indifferent assays, this effect proves a significantly decreased celldivision rate after blockade of GDF-15.

Example 4: B1-23 Inhibits Growth of Tumors In Vivo

The following in vivo study was carried out:

To assess an anti-tumor effect of B1-23 in viva, Balb/c^(nu/nu) nudemice were used in a xenograft setting with the melanoma cell lineUACC-257. The mice were treated either with the antibody B1-23 or withPBS. Each treatment cohort contained 10 Balb/c^(nu/nu) nude mice.

Prior to injection, the UACC-257 melanoma cells were grown in completemedium, excluding any contamination. The cells were harvested when70-80% confluence was reached in the cell culture flask. Cells were thenwashed with PBS and counted. 1×10⁷ viable cells were suspended in PBS.

The first injection/treatment was administered in 6 week oldBalb/c^(nu/nu) nude mice. The inoculation area of the mice was cleanedwith ethanol. The UACC 257 cells were mixed and drawn into a syringewithout a needle, in order to avoid negative pressure on the tumorcells. The cell suspension containing 1×10⁷ cells in PBS was injectedsubcutaneously (s.c.) into the lower flank of the mice.

The intraperitoneal (i.p.) injection of either B1-23 (25 mg/kg bodyweight) or the same volume of PBS started immediately after the tumorcell inoculation (defined as day 1) and was administered twice a week.The tumors were grown for 48 days. The tumor diameters were measuredwith a caliper and the tumor volume in mm³ was calculated by theformula:Volume=(width)²×length/2

The results which were obtained from the study are shown in FIG. 4.

As demonstrated in the Figure, the tumor size of the animal cohorttreated with B1-23 was significantly decreased, compared to the PBScontrol group.

Given that the CDR regions of the mAb B1-23 antibody correspond to CDRregions of the chimeric and humanized antibodies, it is expected thatthe functional properties including anti-cancer effects of theseantibodies are similar.

Example 5: mAb B1-23 Recognizes a Conformational or a DiscontinuousEpitope of Human GDF-15

Epitope Mapping: Monoclonal mouse antibody GDF-15 against 13 mer linearpeptides derived from GDF-15

Antigen: GDF-15: (SEQ ID No: 10)GSGSGSGMPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIGSGSGSG (322 amino acids with linker) 

The protein sequence was translated into 13 mer peptides with a shift ofone amino acid. The C- and N-termini were elongated by a neutral GSGSlinker to avoid truncated peptides (bold letters).

Control Peptides:

Flag: DYKDDDDKGG (SEQ ID No:13), 78 spots; HA: YPYDVPDYAG (SEQ IDNo:14), 78 spots (each array copy)

Peptide Chip Identifier:

000264_01 (10/90, Ala2Asp linker)

Staining Conditions:

Standard buffer: PBS, pH 7.4+0.05% Tween®20

Blocking buffer: Rockland blocking buffer MB-070

Incubation buffer: Standard buffer with 10% Rockland blocking bufferMB-070

Primary sample: Monoclonal mouse antibody GDF-15 (1 μg/μl):

Staining in incubation buffer for 16 h at 4° C. at a dilution of 1:100and slight shaking at 500 rpm

Secondary antibody: Goat anti-mouse IgG (H+L) IRDye680, staining inincubation buffer with a dilution of 1:5000 for 30 min at roomtemperature (RT)

Control antibodies: Monoclonal anti-HA (12CA5)-LL-Attu 680 (1:1000),monoclonal anti-FLAG(M2)-FluoProbes752 (1:1000); staining in incubationbuffer for 1 h at RT

Scanner:

Odyssey® Imaging System, LI-COR Biosciences

Settings: offset: 1 mm; resolution: 21 μm; intensity green/red: 7/7

Results:

After 30 min pre-swelling in standard buffer and 30 min in blockingbuffer, the peptide array with 10, 12 and 15 mer B7H3-derived linearpeptides was incubated with secondary goat anti-mouse IgG (H+L) IRDye680antibody only at a dilution of 1:5000 for 1 h at room temperature toanalyze background interactions of the secondary antibody. ThePEPperCHIP® was washed 2×1 min with standard buffer, rinsed with dist.water and dried in a stream of air. Read-out was done with Odyssey®Imaging System at a resolution of 21 μm and green/red intensities of7/7; We observed a weak interaction of arginine-rich peptides(ELHLRPQAARGRR (SEQ ID No:15), LHLRPQAARGRRR (SEQ ID No:16),HLRPQAARGRRRA (SEQ ID No:17), LRPQAARGRRRAR (SEQ ID No:18),RPQAARGRRRARA (SEQ ID No:19), PQAARGRRRARAR (SEQ ID No:20) andQAARGRRRARARN (SEQ ID No:21)) that are known as frequent binders, andwith the basic peptide MHAQIKTSLHRLK (SEQ ID No:22) due to ionicinteractions with the charged antibody dye.

After pre-swelling for 10 min in standard buffer, the peptide microarraywas incubated overnight at 4° C. with monoclonal mouse antibody GDF-15at a dilution of 1:100. Repeated washing in standard buffer (2×1 min)was followed by incubation for 30 min with the secondary antibody at adilution of 1:5000 at room temperature. After 2×10 sec. washing instandard buffer and short rinsing with dist. water, the PEPperCHIP® wasdried in a stream of air. Read-out was done with Odyssey® Imaging Systemat a resolution of 21 μm and green/red intensities of 7/7 before andafter staining of control peptides by anti-HA and anti-FLAG(M2)antibodies.

It was shown that none of the linear 13 mer peptides derived from GDF-15interacted with monoclonal mouse antibody GDF-15 even at overregulatedintensities. Staining of Flag and HA control peptides that frame thearray, however, gave rise to good and homogeneous spot intensities.

Summary:

The Epitope Mapping of monoclonal mouse GDF-15 antibody against GDF-15did not reveal any linear epitope with the 13 mer peptides derived fromthe antigen. According to this finding it is very likely that monoclonalmouse antibody GDF-recognizes a conformational or a discontinuousepitope with low affinity of partial epitopes. Due to the obviousabsence of any GDF-15 signal above the background staining of thesecondary antibody only, quantification of spot intensities withPepSlide® Analyzer and subsequent peptide annotation were omitted.

Example 6: Structural Identification of Peptide Ligand Epitopes by MassSpectrometric Epitope Excision and Epitope Extraction

The epitope of recombinant human GDF-15 which binds to the antibodyB1-23 was identified by means of the epitope excision method and epitopeextraction method (Suckau et al. Proc Nati Acad Sci USA. 1990 December;87(24): 9848-9852; R. Stefanescu et al., Eur. J. Mass Spectrom. 13,69-75 (2007)).

For preparation of the antibody column, the antibody B1-23 was added toNHS-activated 6-aminohexanoic acid coupled sepharose. Thesepharose-coupled antibody B1-23 was then loaded into a 0.8 mlmicrocolumn and washed with blocking and washing buffers.

Epitope Extraction Experiment:

Recombinant human GDF-15 was digested with trypsin for 2 h at 37° C. (insolution), resulting in different peptides, according to the trypsincleavage sites in the protein. After complete digestion, the peptideswere loaded on the affinity column containing the immobilized antibodyB1-23. Unbound as well as potentially bound peptides of GDF-15 were usedfor mass spectrometry analysis. An identification of peptides by meansof mass spectrometry was not possible. This was a further indicator thatthe binding region of GDF-15 in the immune complex B1-23 comprises adiscontinuous or conformational epitope. In case of a continuous linearepitope, the digested peptides should bind its interaction partner,unless there was a trypsin cleavage site in the epitope peptide. Adiscontinuous or conformational epitope could be confirmed by theepitope excision method described in the following part.

Epitope Excision Experiment:

The immobilized antibody B1-23 on the affinity column was then incubatedwith recombinant GDF-15 for 2 h. The formed immune complex on theaffinity column was then incubated with trypsin for 2 h at 37° C. Thecleavage resulted in different peptides derived from the recombinantGDF-15. The immobilized antibody itself is proteolytically stable. Theresulting peptides of the digested GDF-15 protein, which were shieldedby the antibody and thus protected from proteolytic cleavage, wereeluted under acidic conditions (TFA, pH2), collected and identified bymass spectrometry.

The epitope excision method using MS/MS identification resulted in thefollowing peptides:

Position in Ion/ Peptide sequence Mass Charge EVQVTMCIGACPSQF 40-551769.91 590.50 R (3+) (SEQ ID No: 25) TDTGVSLQTYDDLLA  94-114 2310.96771:33 KDCHCI (3+) (SEQ ID No: 26)

The part of human GDF-15, which binds the antibody B1-23, comprises adiscontinuous or conformational epitope. Mass spectrometry identified 2peptides in the GDF-15 protein, which are responsible for the formationof the immune complex. These peptides are restricted to the positions40-(EVQVTMCIGACPSQFR) and 94-114 (TDTGVSLQTYDDLLAKDCHCI) in the GDF-15amino acid sequence. Thus, these two peptides comprise an epitope of theGDF-15 protein that binds to the antibody B1-23.

Again, since the CDR regions of the mAb B1-23 antibody correspond to CDRregions of the chimeric and humanized antibodies, it is expected thatthe binding properties of these antibodies are similar.

Example 7: Treatment of Cancer-Induced Weight Loss with Anti-GDF-15Antibodies

In the underlying animal study No. 140123, 10 Balb/c^(nu/nu) mice pertreatment group were subcutaneously inoculated with 10×10⁶ UACC-257cells per animal in a 1:1 volume ratio with matrigel (100 μl cells+100μl matrigel). The animals were treated on the same day with therespective antibodies, as indicated below:

Study groups 1-6 Amounts of substances (10 animals per group) (for 45days) 1. Dacarbazine* 80 mg (reference, Lot. No.: C120522C) 2. PBS(SIGMA, Lot. No.: RNBD0341) 30 ml 3. B1-23 anti-GDF-15 antibody 75 mg(murine, Lot. No.: 515980) 4. Chimeric B1-23 anti-GDF-15 antibody 75 mg(chimeric; Lot.: PR0057) 5. H1L5 anti-GDF-15 antibody 75 mg (humanizedB1-23, Lot.: PR3176) 6. B12 Isotype control antibody 75 mg (Isotypeantibody, Lot. No.: ID3195) *Detidemac 500 mg (exp.: March 2015)

The dacarbazine group (group 1) served as a reference group/positivecontrol for tumor growth arrest (cytostatic drug for the treatment ofmalignant melanoma in humans).

The PBS group (group 2) served as a growth control/vehicle controlgroup, because all used substances of the other groups were administeredin PBS.

The group of the murine B1-23 lead candidate antibody (group 3) servedas reference group for a comparison with the chimeric B1-23 antibody andwith the humanized B1-23 H1L5 (groups 4 and 5).

Group 4 is the group of the chimerized B1-23 lead candidate antibody,which contains murine variable domains and constant domains of a humanIgG1 antibody (trastuzumab backbone).

Group 5 is the group of the H1L5 humanized B1-23 lead candidateantibody, which contains humanized frameworks within the murine variableregions and constant domains of a human IgG1 antibody (trastuzumabbackbone).

Group 6 is the group of the B12 isotype antibody. For this isotypecontrol group, the antibody B12 (Lot. No.: ID3195) was produced by thecompany Evitria AG. B12 binds to an HIV antigen and should thereforeneither bind to antigens in nude mice nor to antigens of the humantumor. B12 was selected as a highly suitable isotype control, becausethe immunoglobulin backbone of B12 also consists of the human IgG1antibody trastuzumab and is therefore almost identical to the chimericB1-23 and the H1L5 humanized B1-23 antibodies, except for their variableregions.

The study was carried out in a double-blinded manner for the treatmentwith the antibodies and for the treatment with PBS.

In groups 1, 2 and 6 which did not receive anti-GDF-15 antibodies, morethan 10% body weight loss was observed (i.e. weight loss to a relativebody weight of less than 90% compared to day 0). In contrast, in thegroups which had received treatment with the anti-GDF-15 antibodiesB1-23, chimeric B1-23 and humanized B1-23-H1L5, respectively, anincrease in body weight was observed (FIG. 5).

Thus, surprisingly, treatment with all of the tested anti-GDF-15antibodies completely prevents cancer-induced weight loss in mice. Thiseffect was significant for all of the groups treated with anti-GDF-15antibodies (two-way ANOVA; p<0.05).

It is also noteworthy that the mice of the groups that did not receivetreatment with anti-GDF-15 antibodies exhibited a weight loss of morethan 10%. In humans, a weight loss of as little as 5% over a period of 6months is considered as being indicative of cancer cachexia (Fearon K.et al.: Definition and classification of cancer cachexia: aninternational consensus. Lancet Oncol. 2011 May; 12(5):489-95.). Giventhe larger weight loss of the mice observed in the present study whicheven exceeded 10%, it is expected that the mice in the study, which didnot receive treatment with anti-GDF-15 antibodies, not only exhibitedweight loss but also exhibited cancer cachexia. This effect iscompletely prevented by the anti-GDF-15 antibodies tested. It istherefore expected that the anti-GDF-15 antibodies in accordance withthe invention are capable of both treating cancer-induced weight lossand treating cancer cachexia.

Notably, the extent of weight loss did not correlate with the respectivetumor size (r²=10⁻⁶). If the prevention of weight loss were only asecondary effect resulting from the inhibition of cancer growth and thesmaller tumor sizes, a correlation between tumor size and weight losswould be expected. Thus, the lack of such correlation shows that uses ofthe anti-GDF-15 antibodies according to the invention result in twoindependent treatment effects:

-   -   an inhibition of cancer growth, and    -   a prevention of weight loss as an additional effect, which is        independent from the inhibition of cancer growth, and which is        expected to reflect a prevention of cancer cachexia.

Despite their mechanistic independence, it was observed that theseeffects can occur simultaneously in the same animals.

In addition to evaluating the mean body weight of the mice, the feedconsumption of the mice was evaluated by pairwise comparisons of thestudy groups (Table 1). Notably, the feed consumption of the mice in theanti-GDF-15 antibody groups (B1-23, chimeric B1-23 and humanizedB1-23-H1L5) was significantly higher than the feed consumption of themice in the groups which did not receive the anti-GDF-15 antibodies.

TABLE 1 vs. vs. Feed consumption chimeric humanized. vs. per mouse andday B1-23 B1-23 B1-23 dacarbazine 2.8 ± 0.2 g ** ** ** PBS 2.6 ± 0.4 g** ** ** Chimeric 3.5 ± 0.2 g —— n.s. n.s. B1-23 B12 2.7 ± 0.2 g *** ****** humanized 3.4 ± 0.2 g n.s. —— n.s. B1-23 B1-23 3.6 ± 0.2 g n.s. n.s.—— (* p < 0.05; ** p < 0.01; *** p < 0.001 as assessed by unpairedtwo-sided Student's t-test)

Table 1: Comparative evaluation of the feed consumption between thedifferent treatment groups. For the measured time intervals (day 17-20,day 20-24, day 24-27, day 27-31, day 31-34), the average feedconsumption per mouse and day was calculated for each respective group.The values are indicated together with their standard deviation.

The quality of the humanized anti-GDF-15 antibody B1-23-H1L5 used in thestudy was tested by using gel electrophoresis and coomassie staining ofthe antibodies (see FIG. 6). Notably, the band of the humanizedanti-GDF-15 antibody B1-23-H1L5 was sharp and clear, whereas the bandsof the murine E1-23 anti-GDF-15 antibody and the B12 control antibodyappeared less sharp and at a higher molecular weight. This suggests thatthe humanized anti-GDF-15 antibody B1-23-H1L5 is not prone toaggregation, and that some aggregation may have shifted the molecularweight of the other antibodies to higher values.

Additionally, by using a colorimetric assay, it was confirmed that allanti-GDF-15 antibodies used in the study bound to GDF-15 in aconcentration-dependent manner. To determine the binding of B1-23antibody variants to GDF-15, a colorimetric ELISA experiment wasperformed. The B12 antibody served as an isotype antibody, which doesnot bind to human GDF-15. Therefore, Maxisorp 96 well plates (Nunc) werecoated with hrGDF-15 (25 ng protein per well, 50 μl volume) over nightat 4° C. The following day, plates were washed to remove unbound protein(3 times with 150 μl of PBS 0.05% Tween®) and non-specific binding siteswere blocked with 150 μl of PBS 1% BSA for 2 hours at room temperature.Again, plates were washed and different variants of B1-23 testantibodies were applied (50 μl volume). To inquire specificity of theantibody binding, endpoint dilution was performed starting from 333ng/ml and 1:3 serial dilution. As background control, PBS 1% BSA wasapplied. Following binding for 1 hour at room temperature, wells werewashed as described above. As secondary antibody HRP conjugatedAnti-human IgG (Life technologies, 1:5000) was applied for 1 hour atroom temperature. Wells were washed as described above to remove unboundsecondary antibody. For detection, 50 μl of peroxide substrate (TME1:100 in 0.1 M sodium acetate pH6) were added and following 10 minutesof incubation, 50 μl of stop solution (2N H₂SO₄) were added. As negativecontrols, wells without GDF-15 coating and wells without secondaryantibody were included. For analysis, optical densitiy at 450 nm wasquantified using the ELISA reader (Tecan Sunrise) and the correspondingMagellan software. It was observed that in comparison to the B12antibody, the humanized H1L5 antibody, the chimeric B1-23 antibody andthe murine B1-23 antibody exhibited a clearly concentration-dependentbinding to GDF-15.

Example 8: Determination of Kd Values of Anti-GDF-15 Antibodies

The Kd values of different anti-GDF-15 antibodies were compared usingSurface Acoustic Wave (SAW) gold chip biosensors technology (SAWInstruments GmbH, Schwertberger Str. 16, D-53177 Bonn, Germany):

Antibody: Kd value (nM) B1-23 anti-GDF-15 antibody (murine, IgG2a) 28.8nM Chimeric B1-23 anti-GDF-15 antibody 14 nM (chimerized, human IgG1)H1L5 humanized B1-23 5.62 nM (humanized, human IgG1) Rituxumab (controlantibody) 1116 nM Herceptin (control antibody) No binding

The murine antibody (B1-23) as well as the chimeric B1-23 antibody werepresent in purified form. The H1L5 humanized B1-23 antibody was aserum-free CHO cell culture supernatant. The Kd value of the murineB1-23 deviates from the Kd values determined by Biacore® analyses(surface plasmon resonance) by a factor of 35.

This deviation may—apart from the differences in the measurementmethods—be explained by a reduced availability of free murine B1-23antibody, since it was found that this antibody can form aggregates inits form as mouse antibody. The solution of the murine B1-23 antibodywas therefore stabilized by addition of 0.2% BSA. Therefore, binding ofthe antibody to albumin may have reduced the availability of the murineB1-23 antibody and could explain the differences in the affinity valuesobtained by the different measurement methods. Compared to the murineB1-23 antibody, the H1L5 humanized B1-23 antibody surprisingly showed notendency to aggregate (see also Example 9 below).

In the present assay, the chimeric B1-23 anti-GDF-15 antibody and theH1L5 humanized B1-23 antibody exhibited affinities to human GDF-15 whichwere about 2-fold and 5-fold higher, respectively, than the affinity ofthe murine B1-23 anti-GDF-antibody. Thus, the chimeric B1-23 anti-GDF-15antibody and the H1L5 humanized B1-23 antibody are high affinityantibodies.

Example 9: Aggregation Studies of the Anti-GDF-15 Antibodies

In order to test the aggregation properties of anti-GDF-15 antibodies,antibody samples were shaken for 48 hours at room temperature inmicrocentrifuge tubes, and subsequently, the tubes were visuallyanalyzed for aggregated antibody precipitates.

It was observed that compared to the murine B1-23 antibody, the H1L5humanized B1-23 antibody surprisingly showed no tendency to aggregate,even when the antibody was only present in phosphate-buffered saline(PBS), and when no stabilizing proteins such as BSA were present.

Moreover, in freeze/thaw and dilution experiments, it was observed thatthe H1L5 humanized B1-23 antibody did not aggregate during any of thedilution steps or freeze/thaw cycles.

Additionally, the following experiment was carried out (see FIG. 7):

5 mg of antibody (B1-23, chimeric B1-23=“ChimB1-23”, H1L5) were loadedon Proteus™ protein A columns, eluted and collected in a TRIS Buffer atphysiological pH. After elution, the quality of the purified antibodieswas assessed by Coomassie Brilliant Blue gel analysis. The concentrationof the eluted antibodies was measured photometrically as well as in aBradford assay (Roti-Quant, Carl Roth, Karlsruhe, Germany). All 3antibody solutions (B1-23, ChimB1-23, H1L5) showed similarconcentrations and were adjusted to 0.5 mg/ml. The antibodies were then10 fold concentrated via spin columns (Centricon, MWCO 30). After thisstep, turbidity indicated the presence of precipitates in the samplecontaining B1-23, whereas ChimB1-23 and H1L5 showed no signs ofaggregation. All concentrated eluates were then centrifuged for 5 min at13000 rpm in order to precipitate antibody aggregates. The remainingamount of soluble antibodies was finally determined via Bradford assayfrom the supernatant.

These properties of the antibodies are expected to be advantageous forclinical formulation of the antibodies.

G) INDUSTRIAL APPLICABILITY

The antibodies, antigen-binding portions thereof, pharmaceuticalcompositions and kits according to the present invention may beindustrially manufactured and sold as products for the claimed methodsand uses (e.g. for treating cancer cachexia and cancer), in accordancewith known standards for the manufacture of pharmaceutical products.Accordingly, the present invention is industrially applicable.

PREFERRED EMBODIMENTS

-   -   1. A monoclonal antibody capable of binding to human GDF-15, or        an antigen-binding portion thereof, wherein the heavy chain        variable domain comprises a CDR3 region comprising the amino        acid sequence of SEQ ID NO: 5 or an amino acid sequence at least        90% identical thereto, and wherein the light chain variable        domain comprises a CDR3 region comprising the amino acid        sequence of SEQ ID NO: 7 or an amino acid sequence at least 85%        identical thereto, wherein the constant domain of the heavy        chain comprises the amino acid sequence of SEQ ID No: 29, or an        amino acid sequence at least 85%, preferably at least 90%, more        preferably at least 95% identical thereto, and wherein the        constant domain of the light chain comprises the amino acid        sequence of SEQ ID No: 32, or an amino acid sequence at least        85%, preferably at least 90%, more preferably at least 95%        identical thereto.    -   2. A monoclonal antibody capable of binding to human GDF-15, or        an antigen-binding portion thereof, wherein the binding is        binding to a conformational or discontinuous epitope on human        GDF-15 comprised by the amino acid sequences of SEQ ID No: 25        and SEQ ID No: 26, wherein the constant domain of the heavy        chain comprises the amino acid sequence of SEQ ID No: 29, or an        amino acid sequence at least 85%, preferably at least 90%, more        preferably at least 95% identical thereto, and wherein the        constant domain of the light chain comprises the amino acid        sequence of SEQ ID No: 32, or an amino acid sequence at least        85%, preferably at least 90%, more preferably at least 95%        identical thereto.    -   3. The antibody or an antigen-binding portion thereof of item 1        or 2, wherein the constant domain of the heavy chain comprises        the amino acid sequence of SEQ ID No: 29, or an amino acid        sequence at least 98%, preferably at least 99% identical        thereto, and wherein the constant domain of the light chain        comprises the amino acid sequence of SEQ ID No: 32, or an amino        acid sequence at least 98%, preferably at least 99% identical        thereto.    -   4. The antibody or an antigen-binding portion thereof of any of        items 1 to 3, wherein the constant domain of the heavy chain        comprises the amino acid sequence of SEQ ID No: 29, and wherein        the constant domain of the light chain comprises the amino acid        sequence of SEQ ID No: 32.    -   5. The antibody or antigen-binding portion thereof of any one of        items 1-4, wherein the antibody is a humanized antibody.    -   6. The antibody or antigen-binding portion thereof of item 5,        wherein all of the variable domains of the antibody are        humanized variable domains.    -   7. The antibody or antigen-binding portion thereof of any one of        items 1-6, wherein the heavy chain variable domain comprises the        amino acid sequence of SEQ ID No: 28, or an amino acid sequence        at least 90%, preferably at least 95%, more preferably at least        98%, still more preferably at least 99% identical thereto, and        wherein the light chain variable domain comprises the amino acid        sequence of SEQ ID No: 31, or an amino acid sequence at least        90%, preferably at least 95%, more preferably at least 98%,        still more preferably at least 99% identical thereto.    -   8. The antibody or antigen-binding portion thereof of any one of        items 1-7, wherein the heavy chain variable domain comprises the        amino acid sequence of SEQ ID No: 28, and wherein the light        chain variable domain comprises the amino acid sequence of SEQ        ID No: 31.    -   9. The antibody or antigen-binding portion thereof of any one of        items 1-8, wherein the heavy chain comprises the amino acid        sequence of SEQ ID No: 27, and wherein the light chain comprises        the amino acid sequence of SEQ ID No: 30.    -   10. The antibody or antigen-binding portion thereof of any one        of items 1-4, wherein the heavy chain variable domain comprises        the amino acid sequence of SEQ ID No: 34, or an amino acid        sequence at least 75%, more preferably at least 90%, more        preferably at least 95%, more preferably at least 98%, still        more preferably at least 99% identical thereto, and wherein the        light chain variable domain comprises the amino acid sequence of        SEQ ID No: 37, or an amino acid sequence at least 80%, more        preferably at least 90%, more preferably at least 95%, more        preferably at least 98%, still more preferably at least 99%        identical thereto.    -   11. The antibody or antigen-binding portion thereof of item 10,        wherein the heavy chain variable domain comprises the amino acid        sequence of SEQ ID No: 34, and wherein the light chain variable        domain comprises the amino acid sequence of SEQ ID No: 37.    -   12. The antibody or antigen-binding portion thereof of any one        of items 1-11, wherein the heavy chain variable domain comprises        a CDR1 region comprising the amino acid sequence of SEQ ID NO: 3        and a CDR2 region comprising the amino acid sequence of SEQ ID        NO: 4, and wherein the light chain variable domain comprises a        CDR1 region comprising the amino acid sequence of SEQ ID NO: 6        and a CDR2 region comprising the amino acid sequence        ser-ala-ser.    -   13. The antibody or antigen-binding portion thereof of any one        of items 2-12, wherein the heavy chain variable domain comprises        a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5        or an amino acid sequence at least 90% identical thereto, and        wherein the light chain variable domain comprises a CDR3 region        comprising the amino acid sequence of SEQ ID NO: 7 or an amino        acid sequence at least 85% identical thereto.    -   14. The antibody or antigen-binding portion thereof of any one        of items 1 and 3-12, wherein the binding is binding to a        conformational or discontinuous epitope on human GDF-15 that is        comprised by the amino acid sequences of SEQ ID No: 25 and SEQ        ID No: 26.    -   15. The antibody or antigen-binding portion thereof of any one        of items 1-14, wherein the antibody has a size of more than 100        kDa, preferably more than 110 kDa, more preferably more than 120        kDa, still more preferably more than 130 kDa, and most        preferably more than 140 kDa.    -   16. The antibody or antigen-binding portion thereof of item 15,        wherein the antibody is a full-length antibody.    -   17. The antibody or antigen-binding portion thereof of item 16,        wherein the antibody is a full-length IgG antibody, preferably a        full-length IgG1 antibody.    -   18. The antibody or antigen-binding portion thereof of any one        of items 1 to 17, wherein the antibody has an Fc portion which        is capable of binding to the Fc receptor.    -   19. The antibody or antigen-binding portion thereof of any one        of items 1 to 18, wherein the human GDF-15 is recombinant human        GDF-15 having the amino acid sequence represented by SEQ ID No:        8.    -   20. An antibody or antigen-binding portion thereof according to        any one of items 1 to 19 for use in medicine.    -   21. An antibody or antigen-binding portion thereof according to        any one of items 1 to 19, for use in a method for treating        cancer cachexia in a mammal.    -   22. An antibody or antigen-binding portion thereof according to        any one of items 1 to 19, for use in a method for treating        cancer in a mammal.    -   23. An antibody or antigen-binding portion thereof according to        any one of items 21 to 22 for use according to any one of items        21 to 22, wherein the method is a method for both treating        cancer and treating cancer cachexia in the same mammal.    -   24. The antibody or antigen-binding portion thereof of any one        of items 21 to 23 for the use according to any one of items 21        to 23, wherein the mammal is a human patient.    -   25. The antibody or antigen-binding portion thereof of item 21        or 23-24 for the use according to item 21 or 23-24, wherein the        method for treating cancer cachexia is a method for completely        preventing or completely reverting cancer cachexia.    -   26. The antibody or antigen-binding portion thereof of item 25        for the use according to item 25, wherein the method for        treating cancer cachexia is a method for completely preventing        cancer cachexia.    -   27. The antibody or antigen-binding portion thereof of item 25        for the use according to item 25, wherein the method for        treating cancer cachexia is a method for completely reverting        cancer cachexia.    -   28. The antibody or antigen-binding portion thereof of any one        of items 21₇27 for the use according to any one of items 21-27,        wherein in the method, only mammals suffering from both        -   i) the cancer, and        -   ii) cancer cachexia        -   are treated.    -   29. The antibody or antigen-binding portion thereof of any one        of items 21 or 23-28 for the use according to any one of items        21 or 23-28, wherein the method increases body weight of the        mammal compared to its body weight before the onset of cancer        cachexia.    -   30. The antibody or antigen-binding portion thereof of item 29        for the use according to item 29, wherein the increase in body        weight of the mammal is at least 1.5%, preferably at least 2.5%,        more preferably at least 5% compared to its body weight before        the onset of cancer cachexia.    -   31. The antibody or antigen-binding portion thereof of any one        of items 21 to 30 for the use according to any one of items 21        to 30, wherein the cancer cells of the mammal endogenously        express GDF-15 and/or the cancer cells of the mammal stimulate        endogenous expression of GDF-15 in non-cancerous cells of the        mammal.    -   32. The antibody or antigen-binding portion thereof of any one        of items 21 to 31 for the use according to any one of items 21        to 31, wherein the cancer cells of the mammal endogenously        express GDF-15.    -   33. The antibody or antigen-binding portion thereof of any one        of items 22-32 for the use according to any one of items 22-32,        wherein the method for treating cancer is a method comprising        inhibition of cancer growth.    -   34. The antibody or antigen-binding portion thereof of any one        of items 22-33 for the use according to any one of items 22-33,        wherein the method for treating cancer comprises the induction        of killing of cancer cells by NK cells and CD8+ T cells in the        human patient.    -   35. The antibody or antigen-binding portion thereof of any one        of items 21-34 for the use according to any one of items 21-34,        wherein the human patient has elevated GDF-15 levels in blood        serum before administration.    -   36. The antibody or antigen-binding portion thereof of any one        of items 21-35 for the use according to any one of items 21-35,        wherein the antibody or antigen-binding portion thereof is        -   A) the sole ingredient pharmaceutically active against            cancer used in the method, or        -   B) used in combination with one or more further ingredients            pharmaceutically active against cancer.    -   37. The antibody or antigen-binding portion thereof of any one        of items 21-36 for the use according to any one of items 21-36,        wherein the cancer is selected from the group consisting of        brain cancers including glioma, cancers of the nervous system,        melanoma, lung cancer, lip and oral cavity cancer, hepatic        carcinoma, leukemia, Hodgkin lymphoma, Non-Hodgkin lymphoma,        bladder cancer, cervix uteri cancer, corpus uteri cancer, testis        cancer, thyroid cancer, kidney cancer, gallbladder cancer,        multiple myeloma, nasopharynx cancer, larynx cancer, pharynx        cancer, oesophagus cancer, gastrointestinal tumors including        stomach and colorectal cancer, pancreatic cancer, prostate        cancer, ovarian cancer and breast cancer, preferably from the        group consisting of melanoma, prostate cancer, breast cancer,        brain cancers including glioma, colorectal cancer, stomach        cancer, oesophagus cancer and ovarian cancer, and most        preferably is melanoma.    -   38. The antibody or antigen-binding portion thereof of any one        of items 21-37 for the use according to any one of items 21-37,        wherein prior to administration, the tumor or tumors formed by        the cancer have higher human GDF-15 levels compared to a control        sample of the same patient obtained from a non-cancerous part of        the tissue which is the tissue of origin of the cancer,        preferably 1.2-fold higher levels, more preferably 1.5-fold        higher levels, still more preferably 2-fold higher levels and        most preferably 5-fold higher levels.    -   39. The antibody or antigen-binding portion thereof of item 38        for the use according to item 38, wherein the antibody or        antigen-binding portion thereof is used in combination with one        or more further ingredients pharmaceutically active against        cancer, and wherein the one or more further ingredients        pharmaceutically active against cancer are selected from the        group consisting of: alkylating agents; anti-metabolites;        alkaloids, taxanes; topoisomerase inhibitors; cytotoxic        antibiotics; and radioisotopes.    -   40. The antibody or antigen-binding portion thereof of item 39        for the use according to item 39, wherein the one or more        further ingredients pharmaceutically active against cancer are        selected from the group consisting of: cisplatin, carboplatin,        oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil,        and ifosfamide; azathioprine and mercaptopurine; vincristine,        vinblastine, vinorelbine, and vindesine, paclitaxel, docetaxel,        etoposide and teniposide; irinotecan and topotecan; actinomycin,        anthracyclines, doxorubicin, daunorubicin, valrubicin,        idarubicin, epirubicin, bleomycin, plicamycin and mitomycin.    -   41. A kit comprising the antibody or antigen-binding portion        thereof of any one of items 1-19.    -   42. The kit of item 41 for a use according to any one of items        21 to 40.    -   43. An expression vector comprising a nucleotide sequence        encoding the antibody or antigen-binding portion thereof        according to any of items 1-19.    -   44. A cell line capable of producing an antibody or        antigen-binding portion thereof according to any one of items 1        to 19.    -   45. A monoclonal antibody capable of binding to human GDF-15, or        an antigen-binding portion thereof, wherein the heavy chain        variable domain comprises a CDR3 region comprising the amino        acid sequence of SEQ ID NO: 5 or an amino acid sequence at least        90% identical thereto, and wherein the light chain variable        domain comprises a CDR3 region comprising the amino acid        sequence of SEQ ID NO: 7 or an amino acid sequence at least 85%        identical thereto, for use in a method for treating cancer        cachexia in a mammal.    -   46. A monoclonal antibody capable of binding to human GDF-15, or        an antigen-binding portion thereof, wherein the binding is        binding to a conformational or discontinuous epitope on human        GDF-15 comprised by the amino acid sequences of SEQ ID No: 25        and SEQ ID No: 26, for use in a method for treating cancer        cachexia in a mammal.    -   47. The antibody or antigen-binding portion thereof of item 45        or 46 for the use according to item 45 or 46, wherein the method        for treating cancer cachexia is a method for completely        preventing or completely reverting cancer cachexia.    -   48. The antibody or antigen-binding portion thereof of item 47        for the use according to item 47, wherein the method for        treating cancer cachexia is a method for completely preventing        cancer cachexia.    -   49. The antibody or antigen-binding portion thereof of item 47        for the use according to item 47, wherein the method for        treating cancer cachexia is a method for completely reverting        cancer cachexia.    -   50. The antibody or antigen-binding portion thereof of any one        of items 45-49 for the use according to any one of items 45-49,        wherein in the method, only mammals suffering from both        -   iii) the cancer, and        -   iv) cancer cachexia        -   are treated.    -   51. The antibody or antigen-binding portion thereof of any one        of items 49-50 for the use according to any one of items 49-50,        wherein the method increases body weight of the mammal compared        to its body weight before the onset of cancer cachexia.    -   52. The antibody or antigen-binding portion thereof of item 51        for the use according to item 51, wherein the increase in body        weight of the mammal is at least 1.5%, preferably at least 2.5%,        more preferably at least 5% compared to its body weight before        the onset of cancer cachexia.    -   53. The antibody or antigen-binding portion thereof of any one        of items 45-52 for the use according to any one of items 45-52,        wherein the method is a method for both treating cancer and        treating cancer cachexia in the same mammal.    -   54. The antibody or antigen-binding portion thereof of any one        of items 45-53 for the use according to any one of items 45-53,        wherein the antibody has a size of more than 100 kDa, preferably        more than 110 kDa, more preferably more than 120 kDa, still more        preferably more than 130 kDa, and most preferably more than 140        kDa.    -   55. The antibody or antigen-binding portion thereof of item 54        for the use according to item 54, wherein the antibody is a        full-length antibody.    -   56. The antibody or antigen-binding portion thereof of item 55        for the use according to item 55, wherein the antibody is a        full-length IgG antibody.    -   57. The antibody or antigen-binding portion thereof of any one        of items 45 to 56 for the use according to any one of items 45        to 56, wherein the antibody has an Fc portion which is capable        of binding to the Fc receptor.    -   58. The antibody or antigen-binding portion thereof of any one        of items 45 to 57 for the use according to any one of items 45        to 57, wherein the cancer cells of the mammal endogenously        express GDF-15 and/or the cancer cells of the mammal stimulate        endogenous expression of GDF-15 in non-cancerous cells of the        mammal.    -   59. The antibody or antigen-binding portion thereof of any one        of items 45 to 58 for the use according to any one of items 45        to 58, wherein the cancer cells of the mammal endogenously        express GDF-15.    -   60. The antibody or antigen-binding portion thereof of any one        of items 45 to 59 for the use according to any one of items 45        to 59, wherein the mammal is a human patient.    -   61. The antibody or antigen-binding portion thereof of any one        of items 45 to 60 for the use according to any one of items 45        to 60, wherein the human GDF-15 is recombinant human GDF-15        having the amino acid sequence represented by SEQ ID No: 8.    -   62. The antibody or antigen-binding portion thereof of any one        of items 46-61 for the use according to any one of items 46-61,        wherein the heavy chain variable domain comprises a CDR3 region        comprising the amino acid sequence of SEQ ID NO: 5 or an amino        acid sequence at least 90% identical thereto, and wherein the        light chain variable domain comprises a CDR3 region comprising        the amino acid sequence of SEQ ID NO: 7 or an amino acid        sequence at least 85% identical thereto.    -   63. The antibody or antigen-binding portion thereof of any one        of items 45 and 47-61 for the use according to any one of items        45 and 47-61, wherein the binding is binding to a conformational        or discontinuous epitope on human GDF-15 that is comprised by        the amino acid sequences of SEQ ID No: 25 and SEQ ID No: 26.    -   64. The antibody or antigen-binding portion thereof of any one        of items 53-63 for the use according to any one of items 53-63,        wherein the method for treating cancer is a method comprising        inhibition of cancer growth.    -   65. The antibody or antigen-binding portion thereof of any one        of items 53-64 for the use according to any one of items 53-64,        wherein the method for treating cancer comprises the induction        of killing of cancer cells by NK cells and CD8+ T cells in the        human patient.    -   66. The antibody or antigen-binding portion thereof of any one        of items 45-65 for the use according to any one of items 45-65,        wherein the heavy chain variable domain comprises a CDR1 region        comprising the amino acid sequence of SEQ ID NO: 3 and a CDR2        region comprising the amino acid sequence of SEQ ID NO: 4, and        wherein the light chain variable domain comprises a CDR1 region        comprising the amino acid sequence of SEQ ID NO: 6 and a CDR2        region comprising the amino acid sequence ser-ala-ser.    -   67. The antibody or antigen-binding portion thereof of any one        of items 45-66 for the use according to any one of items 45-66,        wherein the constant domain of the heavy chain comprises the        amino acid sequence of SEQ ID No: 29, or an amino acid sequence        at least 85%, preferably at least 90%, more preferably at least        95% identical thereto, and wherein the constant domain of the        light chain comprises the amino acid sequence of SEQ ID No: 32,        or an amino acid sequence at least 85%, preferably at least 90%,        more preferably at least 95% identical thereto.    -   68. The antibody or antigen-binding portion thereof of any one        of items 45-67 for the use according to any one of items 45-67,        wherein the constant domain of the heavy chain comprises the        amino acid sequence of SEQ ID No: 29, or an amino acid sequence        at least 98%, preferably at least 99% identical thereto, and        wherein the constant domain of the light chain comprises the        amino acid sequence of SEQ ID No: 32, or an amino acid sequence        at least 98%, preferably at least 99% identical thereto.    -   69. The antibody or antigen-binding portion thereof of any one        of items 45-68 for the use according to any one of items 45-68,        wherein the constant domain of the heavy chain comprises the        amino acid sequence of SEQ ID No: 29, and wherein the constant        domain of the light chain comprises the amino acid sequence of        SEQ ID No: 32.    -   70. The antibody or antigen-binding portion thereof of any one        of items 45-69 for the use according to any one of items 45-69,        wherein the antibody is a humanized antibody, and wherein all of        the variable domains of the antibody are humanized variable        domains.    -   71. The antibody or antigen-binding portion thereof of any one        of items 45-70 for the use according to any one of items 45-70,        wherein the heavy chain variable domain comprises the amino acid        sequence of SEQ ID No: 28, or an amino acid sequence at least        90%, preferably at least 95%, more preferably at least 98%,        still more preferably at least 99% identical thereto, and        wherein the light chain variable domain comprises the amino acid        sequence of SEQ ID No: 31, or an amino acid sequence at least        90%, preferably at least 95%, more preferably at least 98%,        still more preferably at least 99% identical thereto.    -   72. The antibody or antigen-binding portion thereof of any one        of items 45-71 for the use according to any one of items 45-71,        wherein the heavy chain variable domain comprises the amino acid        sequence of SEQ ID No: 28, and wherein the light chain variable        domain comprises the amino acid sequence of SEQ ID No: 31.    -   73. The antibody or antigen-binding portion thereof of any one        of items 45-72 for the use according to any one of items 45-72,        wherein the heavy chain comprises the amino acid sequence of SEQ        ID No: 27, and wherein the light chain comprises the amino acid        sequence of SEQ ID No: 30.    -   74. The antibody or antigen-binding portion thereof of any one        of items 45-69 for the use according to any one of items 45-69,        wherein the heavy chain variable domain comprises the amino acid        sequence of SEQ ID No: 34, or an amino acid sequence at least        75%, more preferably at least 90%, more preferably at least 95%,        more preferably at least 98%, still more preferably at least 99%        identical thereto, and wherein the light chain variable domain        comprises the amino acid sequence of SEQ ID No: 37, or an amino        acid sequence at least 80%, more preferably at least 90%, more        preferably at least 95%, more preferably at least 98%, still        more preferably at least 99% identical thereto.    -   75. The antibody or antigen-binding portion thereof of item 74        for the use according to item 74, wherein the heavy chain        variable domain comprises the amino acid sequence of SEQ ID No:        34, and wherein the light chain variable domain comprises the        amino acid sequence of SEQ ID No: 37.    -   76. The antibody or antigen-binding portion thereof of any one        of items 45-66 for the use according to any one of items 45-66,        wherein the antibody is the antibody to human GDF-15 obtainable        from the cell line B1-23 deposited with the Deutsche Sammlung        für Mikroorganismen und Zellkulturen GmbH (DMSZ) under the        accession No. DSM ACC3142 or an antigen-binding portion thereof.    -   77. The antibody or antigen-binding portion thereof of any one        of items 45-75 for the use according to any one of items 45-75,        wherein the heavy chain variable domain comprises a CDR3 region        comprising the amino acid sequence of SEQ ID NO: 5, or wherein        the light chain variable domain comprises a CDR3 region        comprising the amino acid sequence of SEQ ID NO: 7.    -   78. The antibody or antigen-binding portion thereof of any one        of items 45-75 and 77 for the use according to any one of items        45-75 and 77, wherein the heavy chain variable domain comprises        a CDR3 region comprising the amino acid sequence of SEQ ID NO:        5, and wherein the light chain variable domain comprises a CDR3        region comprising the amino acid sequence of SEQ ID NO: 7.    -   79. The antibody or antigen-binding portion thereof of any one        of items 45-69 and 77-78 for the use according to any one of        items 45-69 and 77-78, wherein the heavy chain variable domain        comprises a region comprising an FR1, a CDR1, an FR2, a CDR2 and        an FR3 region and comprising the amino acid sequence of SEQ ID        NO: 1 or a sequence 95% identical thereto, and wherein the light        chain variable domain comprises a region comprising an FR1, a        CDR1, an FR2, a CDR2 and an FR3 region and comprising the amino        acid sequence of SEQ ID NO: 2 or a sequence 95% identical        thereto.    -   80. The antibody or antigen-binding portion thereof of any one        of items 45-69 and 77-79 for the use according to any one of        items 45-69 and 77-79, wherein the heavy chain variable domain        comprises a region comprising an FR1, a CDR1, an FR2, a CDR2 and        an FR3 region and comprising the amino acid sequence of SEQ ID        NO: 1 or a sequence 98% identical thereto, and wherein the light        chain variable domain comprises a region comprising an FR1, a        CDR1, an FR2, a CDR2 and an FR3 region and comprising the amino        acid sequence of SEQ ID NO: 2 or a sequence 98% identical        thereto.    -   81. The antibody or antigen-binding portion thereof of any one        of items 45-80 for the use according to any one of items 45-80,        wherein the antibody or antigen-binding portion thereof has an        equilibrium dissociation constant for human GDF-15 that is equal        to or less than 20 nM, preferably less than 10 nM, more        preferably less than 5 nM and most preferably between 0.1 nM and        2 nM.    -   82. The antibody or antigen-binding portion thereof of any one        of items 45-75 and 77-81 for the use according to any one of        items 45-75 and 77-81, wherein the antibody or antigen-binding        portion thereof binds to the same human GDF-15 epitope as the        antibody to human GDF-15 obtainable from the cell line B1-23        deposited with the Deutsche Sammlung für Mikroorganismen und        Zellkulturen GmbH (DMSZ) under the accession No. DSM ACC3142.    -   83. The antibody or antigen-binding portion thereof of any one        of items 45-82 for the use according to any one of items 45-82,        wherein the human patient has elevated GDF-15 levels in blood        serum before administration.    -   84. The antibody or antigen-binding portion thereof of any one        of items 45-83 for the use according to any one of items 45-83,        wherein the antibody or antigen-binding portion thereof is        -   A) the sole ingredient pharmaceutically active against            cancer used in the method, or        -   B) used in combination with one or more further ingredients            pharmaceutically active against cancer.    -   85. The antibody or antigen-binding portion thereof of any one        of items 45-84 for the use according to any one of items 45-84,        wherein the cancer is selected from the group consisting of        brain cancers including glioma, cancers of the nervous system,        melanoma, lung cancer, lip and oral cavity cancer, hepatic        carcinoma, leukemia, Hodgkin lymphoma, Non-Hodgkin lymphoma,        bladder cancer, cervix uteri cancer, corpus uteri cancer, testis        cancer, thyroid cancer, kidney cancer, gallbladder cancer,        multiple myeloma, nasopharynx cancer, larynx cancer, pharynx        cancer, oesophagus cancer, gastrointestinal tumors including        stomach and colorectal cancer, pancreatic cancer, prostate        cancer, ovarian cancer and breast cancer, preferably from the        group consisting of melanoma, prostate cancer, breast cancer,        brain cancers including glioma, colorectal cancer, stomach        cancer, oesophagus cancer and ovarian cancer, and most        preferably is melanoma.    -   86. The antibody or antigen-binding portion thereof of any one        of items 45-85 for the use according to any one of items 45-85,        wherein prior to administration, the tumor or tumors formed by        the cancer have higher human GDF-15 levels compared to a control        sample of the same patient obtained from a non-cancerous part of        the tissue which is the tissue of origin of the cancer,        preferably 1.2-fold higher levels, more preferably 1.5-fold        higher levels, still more preferably 2-fold higher levels and        most preferably 5-fold higher levels.    -   87. The antibody or antigen-binding portion thereof of item 84        for the use according to item 84, wherein the antibody or        antigen-binding portion thereof is used in combination with one        or more further ingredients pharmaceutically active against        cancer, and wherein the one or more further ingredients        pharmaceutically active against cancer are selected from the        group consisting of: alkylating agents; anti-metabolites;        alkaloids, taxanes; topoisomerase inhibitors; cytotoxic        antibiotics; and radioisotopes.    -   88. The antibody or antigen-binding portion thereof of item 87        for the use according to item 87, wherein the one or more        further ingredients pharmaceutically active against cancer are        selected from the group consisting of: cisplatin, carboplatin,        oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil,        and ifosfamide; azathioprine and mercaptopurine; vincristine,        vinblastine, vinorelbine, and vindesine, paclitaxel, docetaxel,        etoposide and teniposide; irinotecan and topotecan; actinomycin,        anthracyclines, doxorubicin, daunorubicin, valrubicin,        idarubicin, epirubicin, bleomycin, plicamycin and mitomycin.

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The invention claimed is:
 1. A monoclonal antibody that specificallybinds to human GDF-15, or an antigen-binding portion thereof,comprising: a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 28; and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO:
 31. 2. Amonoclonal antibody that specifically binds to human GDF-15, or anantigen-binding portion thereof, comprising a heavy chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:
 28. 3.A monoclonal antibody that specifically binds to human GDF-15, or anantigen-binding portion thereof, comprising a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO:
 31. 4.A monoclonal antibody that specifically binds to human GDF-15, or anantigen-binding portion thereof, comprising: a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO: 27; and a light chaincomprising the amino acid sequence set forth in SEQ ID NO:
 30. 5. Amonoclonal antibody that specifically binds to human GDF-15, or anantigen-binding portion thereof, comprising a heavy chain comprising theamino acid sequence set forth in SEQ ID NO:
 27. 6. A monoclonal antibodythat specifically binds to human GDF-15, or an antigen-binding portionthereof, comprising a light chain comprising the amino acid sequence setforth in SEQ ID NO:
 30. 7. The monoclonal antibody or antigen-bindingportion thereof of claim 1, comprising a human heavy chain constantregion.
 8. The monoclonal antibody or antigen-binding portion thereof ofclaim 1, comprising a human IgG1 or IgG4 heavy chain constant region. 9.The monoclonal antibody or antigen-binding portion thereof of claim 1,comprising a human light chain constant region.
 10. The monoclonalantibody or antigen-binding portion thereof of claim 1, comprising ahuman kappa light chain constant region.
 11. A pharmaceuticalcomposition comprising the antibody or antigen-binding portion thereofof claim
 1. 12. A pharmaceutical composition comprising the antibody orantigen-binding portion thereof of claim
 4. 13. A nucleotide sequenceencoding the heavy chain variable domain and/or the light chain variabledomain of the antibody of claim
 1. 14. An expression vector comprisingthe nucleotide sequence of claim
 13. 15. A cell line comprising theexpression vector of claim
 14. 16. A nucleotide sequence encoding theheavy chain and/or the light chain of the antibody of claim
 4. 17. Anexpression vector comprising the nucleotide sequence of claim
 16. 18. Acell line comprising the expression vector of claim 17.